Anhydrous Crystalline Forms of N-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide

ABSTRACT

The invention comprises (1) anhydrous crystalline forms of N-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide, (2) pharmaceutical compositions comprising at least one such form, (3) methods for the treatment of a phosphodiesterase-5-mediated condition using at least one such form, and (4) methods for preparing such forms. The compound N-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide has the following structure (I).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/680,445 filed May 12, 2005 and U.S. Provisional Application Ser.No. 60/681,711 filed May 17, 2005, the disclosure of each of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to crystalline forms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.More specifically, this invention relates to (1) anhydrous crystallineforms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,(2) pharmaceutical compositions comprising at least one such form, (3)methods for the treatment of a phosphodiesterase-5-mediated conditionusing at least one such form, and (4) methods for preparing such forms.

BACKGROUND OF THE INVENTION

The compoundN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidehas the following structure (1):

The synthesis ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideis described in Example 115 of published PCT application WO 2005/049616(the “Compound Application”). The Compound Application further disclosesthatN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideis a phosphodiesterase-5 (“PDE-5”) inhibitor that can be used to treat aPDE-5-mediated condition, such as hypertension.

Different solid-state forms of a pharmaceutical compound can havematerially different physical properties. Such differences in physicalproperties can have an impact, for example, on how a pharmaceuticalcompound is made, processed, formulated or administered. Accordingly,the identification of new solid-state forms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidethat provide an advantage relative to other solid-state forms in making,processing, formulating or administering the compound are desirable. Asdiscussed below, three new anhydrous crystalline forms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidehave been identified.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to anhydrouscrystalline forms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

In another embodiment, the invention is directed to the Form A anhydrouscrystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide(“Form A”).

In another embodiment, the invention is directed to the Form B anhydrouscrystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide(“Form B”).

In another embodiment, the invention is directed to the Form C anhydrouscrystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide(“Form C”).

In another embodiment, the invention is directed to a compositioncomprising at least two forms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideselected from the group consisting of Form A, Form B, and Form C.

In another embodiment, the invention is directed to a pharmaceuticalcomposition comprising at least one form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideselected from the group consisting of Form A, Form B, and Form C and apharmaceutically acceptable carrier.

In another embodiment, the invention is directed to methods for thetreatment of a PDE-5-mediated condition comprising administering to asubject a therapeutically-effective amount at least one form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideselected from the group consisting of Form A, Form B, and Form C.

In another embodiment, the invention is directed to methods for thepreparation of Form A, Form B, and Form C.

Additional embodiments of the invention are discussed throughout thespecification of this application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustrative X-ray powder diffraction pattern for FormA.

FIG. 2 shows a calculated X-ray powder diffraction pattern for Form A.

FIG. 3 shows an illustrative X-ray powder diffraction pattern for FormB.

FIG. 4 shows an illustrative X-ray powder diffraction pattern for FormC.

FIG. 5 shows an illustrative DSC thermogram for Form A.

FIG. 6 shows an illustrative DSC thermogram for Form B.

FIG. 7 shows an illustrative DSC thermogram for Form C.

FIG. 8 shows an illustrative FT-IR spectrum for Form A.

FIG. 9 shows an illustrative FT-IR spectrum for Form B.

FIG. 10 shows an illustrative FT-IR spectrum for Form C.

FIG. 11 shows an illustrative Raman spectrum for Form A.

FIG. 12 shows an illustrative Raman spectrum for Form B.

FIG. 13 shows an illustrative Raman spectrum for Form C.

FIG. 14 shows an X-ray powder diffraction pattern for the materialprepared in Example 1.

FIG. 15 shows an illustrative alternative synthetic scheme for thepreparation ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The solid-state form of a compound can materially affect the physicalproperties of the compound including: (1) packing properties such asmolar volume, density and hygroscopicity, (2) thermodynamic propertiessuch as melting temperature, vapor pressure and solubility, (3) kineticproperties such as dissolution rate and stability (including stabilityat ambient conditions, especially to moisture and under storageconditions), (4) surface properties such as surface area, wettability,interfacial tension and shape, (5) mechanical properties such ashardness, tensile strength, compactability, handling, flow and blend; or(6) filtration properties. Selection and control of the solid-state formis particularly important for compounds that are pharmacological agents.Careful selection and control of the solid-state form can reducesynthesis, processing, formulation or administration problems associatedwith the compound.

Three new anhydrous crystalline forms (Form A, Form B and Form C) of thecompoundN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidehave been identified. As explained in greater detail below, Form A, FormB, and Form C each have distinct physical properties relative to eachother.

As used in this application, the nomenclature“N-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide”(as well as the corresponding “structure 1”) is intended to embrace alltautomeric isomers ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.For example, two tautomeric isomers ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideare shown below as Tautomer (1) and Tautomer (2) (exemplified by theresonance structures below):

Without being held to a particular theory, it is hypothesized that FormA crystallizes as Tautomer (1) above, and that Form B and Form C eachcrystallize as Tautomer (2) above.

A. Abbreviations and Definitions

As used in reference to ¹H NMR, the symbol “δ” refers to a ¹H NMRchemical shift.

As used in reference to ¹H NMR, the abbreviation “br” refers to a broad¹H NMR signal.

As used in reference to ¹H NMR, the abbreviation “d” refers to a doublet¹H NMR peak.

The abbreviation “m/z” refers to a Mass spectrum peak.

As used in reference to ¹H NMR, the abbreviation “m” refers to amultiplet ¹H NMR peak.

As used in reference to ¹H NMR, the abbreviation “q” refers to a quartet¹H NMR peak.

As used in reference to ¹H NMR, the abbreviation “s” refers to a singlet¹H NMR peak.

As used in reference to ¹H NMR, the abbreviation “t” refers to a triplet¹H NMR peak.

The term “DSC” refers to differential scanning calorimetry.

The term “HPLC” refers to high pressure liquid chromatography.

The term “PXRD” refers to X-ray powder diffraction

The terms “PDE-5-mediated condition” and “phosphodiesterase-5-mediatedcondition” refer to any condition mediated by PDE-5, whether throughdirect regulation by PDE-5, or through indirect regulation by PDE-5 as acomponent of a signaling pathway.

The term “composition” refers to an article of manufacture which resultsfrom the mixing or combining of more than one element or ingredient.

The term “crystalline form” as applied toN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamiderefers to a solid-state form wherein theN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidemolecules are arranged to form a distinguishable crystal lattice (i)comprising distinguishable unit cells, and (ii) yielding diffractionpeaks when subjected to X-ray radiation.

The term “crystallization” as used throughout this application can referto crystallization and/or recrystallization depending upon theapplicable circumstances relating to the preparation of theN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidestarting material.

The term “purity” refers to the chemical purity ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideaccording to conventional HPLC assay.

The term “phase purity” refers to the solid-state purity ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewith regard to a particular solid-state form of theN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideas determined by the analytical methods described herein.

The term “pharmaceutically acceptable carrier” refers to a carrier thatis compatible with the other ingredients of the composition and is notdeleterious to the subject. Such carriers may be pharmaceuticallyacceptable material, composition or vehicle, such as a liquid or solidfiller, diluent, excipient, solvent or encapsulating material, involvedin carrying or transporting a chemical agent. The preferred compositiondepends on the method of administration.

The terms “prevent,” “prevention” or “preventing” refer to eitherpreventing the onset of a preclinically evident condition altogether orpreventing the onset of a preclinical evident stage of a condition in asubject. Prevention includes, but is not limited to, prophylactictreatment of a subject at risk of developing a condition.

The term “relative intensity” refers to the ratio of the intensity of anindividual diffraction peak (or spectral line as the case may be) to theintensity of the strongest diffraction peak in the same diffractionpattern. In other words, the intensity of the strongest peak is set to100 and all other intensities are scaled accordingly.

The term “therapeutically effective amount” refers to that amount ofdrug or pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system or animal that is being sought by aresearcher or clinician.

The term “treatment” (and corresponding terms “treat” and “treating”)refers to palliative, restorative, and preventative treatment of asubject. The term “palliative treatment” refers to treatment that easesor reduces the effect or intensity of a condition in a subject withoutcuring the condition. The term “preventative treatment” (and thecorresponding term “prophylactic treatment”) refers to treatment thatprevents the occurrence of a condition in a subject. The term“restorative treatment” refers to treatment that halts the progressionof, reduces the pathologic manifestations of, or entirely eliminates acondition in a subject.

B. Characterization of Crystalline Forms

The crystalline state of a compound can be described by severalcrystallographic parameters, including single crystal structure, X-raypowder diffraction pattern, melting temperature, infrared absorptionspectroscopy pattern, and Raman spectroscopy pattern.

1. Single Crystal X-Ray Analysis

The crystal structure of Form A was determined by single crystal X-raydiffraction analysis. The single crystal X-ray diffraction data used inthe analysis were collected at room temperature using a Bruker SMARTAPEX Single Crystal X-Ray diffractometer and Mo Kα radiation.Intensities were integrated (SMART v5.622 (control) and SAINT v6.02(integration) software, Bruker AXS Inc., Madison, Wis. 1994) fromseveral series of exposures where each exposure covered 0.3° in ω, withan exposure time of 30 seconds and the total data set was more than ahemisphere. Data were corrected for absorption using the multiscansmethod (SADABS, Program for scaling and correction of area detectordata, G. M. Sheldrick, University of Göttingen, 1997 (based on themethod of R. H. Blessing, Acta Cryst. 1995, A51, 33-38)). The crystalstructure was then solved by direct methods using SHELXS-97 (Program forcrystal structure refinement. G. M. Sheldrick, University of Göttingen,Germany, 1997, release 97-2), in Space Group P2₁/c and refined by themethod of least-squares using SHELXL-97. Selected crystal structure dataare summarized in Table 1A.

The crystal structure of Form C was also determined by single crystalX-ray diffraction analysis in the same manner as described above forForm A except that an exposure time of 120 seconds was used. The crystalstructure was solved by direct methods using SHELXS-97, in Space GroupP-1, and refined by the method of least-squares using SHELXL-97.Selected crystal structure data for Form C are summarized in Table 1B.

TABLE 1A Form A Crystal Structure Data Parameter Value Crystal SystemMonoclinic Space Group P2 ₁/c a  12.9809(11) Å b  18.1064(15) Å c 21.0685(17) Å alpha  90° beta  98.832(2)° gamma  90° Wavelength  0.71073 Å Volume 4893.2(7) Å³ Z   8 Density (calculated)   1.294 Mg/m³

TABLE 1B Form C Crystal Structure Data Parameter Value Crystal SystemTriclinic Space Group P-1 a   6.935(4) Å b  12.734(7) Å c  13.350(7) Åalpha  100.252(9)° beta  91.272(11)° gamma  95.544(10)° Wavelength  0.71073 Å Volume 1153.8(11) Å³ Z   2 Density (calculated)   1.372Mg/m³

As previously noted, it is hypothesized that Form A crystallizes asTautomer (1) and Form C crystallizes as Tautomer (2). Single crystalX-ray analysis supports this hypothesis.

2. X-Ray Powder Diffraction

The crystal structures of Form A, Form B and Form C were analyzed usingX-ray powder diffraction (“PXRD”). The X-ray diffraction data werecollected at room temperature using a Bruker AXS D4 powder X-raydiffractometer (Cu Kα radiation) fitted with an automatic samplechanger, a theta-theta goniometer, automatic beam divergence slits, asecondary monochromator and a scintillation counter. Samples wereprepared for analysis by packing the powder into a 12 mm diameter, 0.25mm deep cavity that had been cut into a silicon wafer specimen mount.The sample was rotated while being irradiated with copper K-alpha₁X-rays (wavelength=1.5406 Ångstroms) with the X-ray tube operated at 40kV/40 mA. The analyses were performed with the goniometer running incontinuous mode set for a 5 second count per 0.02° step over a two thetarange of 2° to 55°. The peaks obtained for Form A were aligned againstthose from the calculated pattern from the single crystal structure. ForForm B and Form C, the peaks obtained were aligned against a siliconreference standard.

For Form A, 2-theta angles, d spacings, and relative intensities werecalculated from the single crystal structure using the “Reflex PowderDiffraction” module of Accelrys Materials Studio™ [version 2.2].Pertinent simulation parameters were in each case: Wavelength=1.540562 Å(Cu K-alpha₁), Polarisation Factor=0.5; and Pseudo-Voigt Profile(U=0.01, V=−0.001, W=0.002).

As will be appreciated by the skilled crystallographer, the relativeintensities of the various peaks reported in the Tables and Figuresbelow may vary due to a number of factors such as orientation effects ofcrystals in the X-ray beam or the purity of the material being analyzedor the degree of crystallinity of the sample. The peak positions mayalso shift for variations in sample height but the peak positions willremain substantially as defined in Tables 2A, 2C, and 2D for Form A,Form B and Form C, respectively. The skilled crystallographer also willappreciate that measurements using a different wavelength will result indifferent shifts according to the Bragg equation −nλ=2d sin θ, Suchfurther PXRD patterns generated by use of alternative wavelengths areconsidered to be alternative representations of the PXRD patterns of thecrystalline materials of the present invention and as such are withinthe scope of the present invention.

Illustrative PXRD patterns for Form A, Form B and Form C are shown inFIGS. 1, 3 and 4, respectively. Tables 2A, 2C and 2D list thecorresponding main diffraction peaks in terms of 2θ values andintensities for Form A, Form B and Form C, respectively. Table 2A liststhe Form A peaks having a relative intensity greater than 25%. Table 2Clists the Form B peaks having a relative intensity greater than 2%.Table 2D lists the Form C peaks having a relative intensity greater than10%.

In addition, a calculated PXRD pattern for Form A is shown in FIG. 2.Table 2B lists the corresponding calculated main diffraction peaks interms of 2θ values and intensities for Form A. Table 2B lists thecalculated Form A peaks having a relative intensity greater than 10%.

TABLE 2A Form A PXRD Data Angle Relative Angle Relative 2-ThetaIntensity 2-Theta Intensity (degrees) (%) (degrees) (%) 8.5 84.3 22.737.9 9.0 55.8 23.5 59.2 14.0 25.3 23.9 25.2 16.9 68.5 24.8 47.7 18.242.9 25.0 37.0 19.9 100.0 25.4 65.8 21.0 58.9 26.0 42.9 21.4 55.0 26.241.3 21.7 39.4 30.3 36.0 22.5 99.1 33.9 27.2

TABLE 2B Form A Calculated PXRD Data Angle Relative Angle Relative2-Theta Intensity 2-Theta Intensity (degrees) (%) (degrees (%) 8.5 100.020.0 22.7 9.0 57.9 21.0 20.3 9.9 14.5 21.4 14.0 13.0 10.2 21.7 10.7 14.112.6 22.5 30.2 16.9 22.2 23.6 13.9 17.0 12.0 24.8 14.2 18.3 17.3 25.513.7 19.9 15.1 26.3 12.3

TABLE 2C Form B PXRD Data Angle Relative Angle Relative 2-ThetaIntensity 2-Theta Intensity (degrees) (%) (degrees) (%) 3.6 47.3 19.43.2 7.2 100.0 21.8 2.7 9.4 2.9 22.9 2.5 10.1 4.1 23.8 7.6 14.4 5.8 27.03.6 18.1 2.2 29.1 4.1 18.9 2.2 32.9 3.5 19.3 3.8

TABLE 2D Form C PXRD Data Angle Relative Angle Relative 2-ThetaIntensity 2-Theta Intensity (Degrees) % (Degrees) % 6.7 100.0 20.2 31.27.1 29.9 21.4 10.5 10.6 57.4 23.1 13.8 12.8 7.3 23.8 7.7 14.0 27.1 25.811.2 14.5 7.4 26.1 23.1 14.8 6.3 27.0 11.9 15.9 7.3 27.2 17.7 16.8 7.132.3 14.9 17.7 69.3 33.6 16.0 19.1 8.2 34.2 16.7

Form A PXRD

Form A has a PXRD pattern that comprises at least one diffraction peakselected from the group consisting of 8.5*0.1; 9.0±0.1; 16.9±0.1;20.0±0.1; and 22.5±0.1 degrees two theta. In one embodiment, Form A hasa PXRD pattern that comprises a diffraction peak at 8.5±0.1 degrees twotheta. In another embodiment, Form A has a PXRD pattern that comprises adiffraction peak at 8.5±0.1 degrees two theta, and further comprises atleast one additional diffraction peak selected from the group consistingof 9.0±0.1; 16.9±0.1; 20.0±0.1; and 22.5±0.1 degrees two theta. Inanother embodiment, Form A has a PXRD pattern that comprises diffractionpeaks at 8.5±0.1; 9.0±0.1; and 16.9±0.1 degrees two theta. In anotherembodiment, Form A has a PXRD pattern that comprises diffraction peaksat 8.5±0.1; 9.0±0.1; 16.9±0.1; 20.0±0.1; and 22.5±0.1 degrees two theta.In the above embodiments, the diffraction peaks identified at 8.5±0.1;9.0±0.1; 16.9±0.1; 20.0±0.1; and 22.5±0.1 degrees two theta typicallyhave a relative intensity of at least about 10%.

In another embodiment, Form A has a PXRD pattern that (a) comprises atleast one diffraction peak selected from the group consisting of8.5±0.1; 9.0±0.1; 16.9±0.1; 20.0±0.1; and 22.5±0.1 degrees two theta,and (b) does not comprise at least one diffraction peak selected fromthe group consisting of 3.6±0.1 and 7.2±0.1 degrees two theta.

Form B PXRD

Form B has a PXRD pattern that comprises at least one diffraction peakselected from the group consisting of 3.6±0.1; 7.2±0.1, 10.1±0.1,14.4±0.1; and 23.8±0.1 degrees two theta. In one embodiment, Form B hasa PXRD pattern that comprises a diffraction peak at 3.6±0.1 degrees twotheta. In another embodiment, Form B has a PXRD pattern that comprises adiffraction peak at 3.6±0.1 degrees two theta, and further comprises atleast one additional diffraction peak selected from the group consistingof 7.2±0.1, 10.1±0.1, 14.4±0.1 and 23.8±0.1 degrees two theta. Inanother embodiment, Form B has a PXRD pattern that comprises diffractionpeaks at 3.6±0.1 and 7.2±0.1 degrees two theta. In another embodiment,Form B has a PXRD pattern that comprises diffraction peaks at 3.6±0.1;7.2±0.1; and 23.8±0.1 degrees two theta. In another embodiment, Form Bhas a PXRD pattern that comprises diffraction peaks at 3.6±0.1; 7.2±0.1;10.1±0.1; 14.4±0.1; and 23.8±0.1 degrees two theta. In the aboveembodiments, the diffraction peaks identified at 3.6±0.1 and 7.2±0.1degrees two theta typically have a relative intensity of at least about10%.

In another embodiment, Form B has a PXRD pattern that (a) comprises atleast one diffraction peak selected from the group consisting of3.6±0.1; 7.2±0.1, 10.1±0.1, 14.4±0.1; and 23.8±0.1 degrees two theta,and (b) does not comprise at least one diffraction peak selected fromthe group consisting of 8.5±0.1; 6.7±0.1; and 22.5±0.1 degrees twotheta.

Form C PXRD

Form C has a PXRD pattern that comprises at least one diffraction peakselected from the group consisting of and 6.7±01, 10.6±0.1; 14.0±0.1;17.7±0.1; and 20.2±0.1 degrees two theta. In one embodiment, Form C hasa PXRD pattern that comprises a diffraction peak at 6.7±0.1 degrees twotheta. In one embodiment, Form C has a PXRD pattern that comprises adiffraction peak at 10.6±0.1 degrees two theta. In one embodiment, FormC has a PXRD pattern that comprises a diffraction peak at 14.0±0.1degrees two theta. In one embodiment, Form C has a PXRD pattern thatcomprises a diffraction peak at 17.7±0.1 degrees two theta. In oneembodiment, Form C has a PXRD pattern that comprises a diffraction peakat 20.2±0.1 degrees two theta. In another embodiment, Form C has a PXRDpattern that comprises a diffraction peak at 6.7±0.1 degrees two theta,and further comprises at least one additional diffraction peak selectedfrom the group consisting 10.6±0.1; 14.0±0.1; 17.7±0.1; and 20.21±0.1degrees two theta. In another embodiment, Form C has a PXRD pattern thatcomprises diffraction peaks at 6.7±0.1 and 20.2±0.1 degrees two theta.In another embodiment, Form C has a PXRD pattern that comprisesdiffraction peaks at 6.7±0.1; 17.7±0.1; and 20.2±0.1 degrees two theta.In another embodiment, Form C has a PXRD pattern that comprisesdiffraction peaks at 6.7±0.1; 17.7±0.1; 10.6±0.1; and 20.2±0.1 degreestwo theta. In another embodiment, Form C has a PXRD pattern thatcomprises diffraction peaks at 6.7±0.1; 10.6±0.1; 14.0±0.1; 17.7±0.1;and 20.2±01 degrees two theta. In the above embodiments, the diffractionpeaks identified at 6.7±0.1; 10.6±0.1; 14.0±0.1; 17.7±0.1; and 20.2±0.1degrees two theta preferably have a relative intensity of at least about10%.

In another embodiment, Form C has a PXRD pattern that (a) comprises atleast one diffraction peak selected from the group consisting of6.7±0.1; 10.6±0.1; 14.0±0.1; 17.7±0.1; and 20.2±0.1 degrees two theta,and (b) does not comprise at least one diffraction peak selected fromthe group consisting of 3.6±0.1 and 9.0±0.1 degrees two theta.

3. Differential Scanning Calorimetry

Form A, Form B and Form C were each analyzed using differential scanningcalorimetry (DSC). A TA Instruments Q1000 differential scanningcalorimeter was used to perform each analysis. Each sample was heatedfrom 25 to 300° C. at 20° C. per minute in an aluminium pan with the lidlaid on top, with a nitrogen purge gas. The temperature of the meltingendothermic peak was reported as the melting point. The data from DSCanalyses are dependent on several factors, including the rate ofheating, the purity of the sample, crystal size, and sample size.Therefore, the following melting points are representative of thesamples as prepared below.

Form A DSC

A 3.171 mg sample of Form A was analyzed by DSC as described above. TheDSC thermogram obtained for the sample of Form A is shown in FIG. 5.Form A shows a first endothermic peak at 174° C.±3° C., followed by anexothermic recrystallization event at 179° C.±3° C., and a secondendothermic peak at 219° C.±3° C. The peak at 174° C.±3° C. correspondsto the melting of Form A. The exothermic recrystallisation event at 179°C.±3° C. corresponds to the recrystallization of the melted compound asForm B. The peak at 219° C.±3° C. corresponds to the melting of Form B.

Form B DSC

A 1.603 mg sample of Form B was analyzed by DSC as described above. TheDSC thermogram for obtained for the sample of Form B is shown in FIG. 6.Form B shows an endothermic peak at 218° C.±3° C. that corresponds tothe melting of Form B.

Form C DSC

A 4.405 mg sample of Form C was analyzed by DSC as described above. TheDSC thermogram for obtained for the sample of Form C is shown in FIG. 7.Form C shows a first endothermic peak at 188° C.±3° C., followed by anexothermic recrystallization event at 199° C.±3° C., and a secondendotherm at 219° C.±3° C. The peak at 188° C.±3° C. corresponds to themelting of Form C. The exothermic recrystallisation event at 199° C.±3°C. corresponds to the recrystallization of the melted compound as FormB. The peak at 219° C.±3° C. corresponds to the melting of Form B.

4. Fourier-Transform Infrared Spectroscopy

The crystal structures of Form A, Form B and Form C were analyzed usingFourier-Transform infrared (“FT-IR”) spectroscopy. FT-IR spectra forsamples of Form A, Form B and Form C were obtained using a ThermoNicoletAvatar 360 spectrometer with a Smart Golden Gate single reflection ATRaccessory (diamond top-plate and zinc-selenide lenses). The measurementswere collected using 2 cm−1 resolution, 128 scans, and Happ Genzelapodization. Because the FT-IR spectra were recorded using singlereflection ATR, no sample preparation was required. Using ATR FT-IR,however, will cause the relative intensities of infrared bands to differfrom those typically seen in a KBr disc FT-IR spectrum. Due to thenature of ATR FT-IR, band intensities generally increase when going fromthe higher wavenumber region to the lower wavenumber region.Experimental error, unless otherwise noted, was ±2 cm−1.

Illustrative FT-IR spectra for Form A, Form B and Form C are shown inFIGS. 8, 9 and 10, respectively. Tables 4A, 4B and 4C list thecorresponding unique and assignable absorption bands for Form A, Form Band Form C, respectively.

TABLE 4A Form A FT-IR Spectroscopy Data Absorption Band¹ FunctionalGroup 3247 m², 3201 m NH stretch (amine and amide) 1707 s C═O stretch(amide) Region 1603-1524 C═C, C═N ring stretch and C—N—H (1603 s, 1573m, bend (amide II/sulphonamide 1540 m) and amine) 1334 m, 1325 w, 1314 wSO₂ asymmetric stretch 1188 m 1120 m 1085 w  998 w  933 w  928 w  831 m 810 m  775 m  696 m ¹w: weak; m: medium; ms: medium-strong; s: strong²Experimental error was ±3 cm⁻¹.

TABLE 4B Form B FT-IR Spectroscopy Data Absorption Band¹ FunctionalGroup 1704 s C═O stretch (amide) 1646 s Acyclic C═N stretch Region1599-1530 C═C, C═N ring stretch and C—N—H (1599 w, 1577 s, bend (amideII/sulphonamide 1530 m) and amine) 1452 m 1395 m 1338 m, 1321 m SO₂asymmetric stretch 1211 w 1112 s  920 w  781 ms  722 m  688 m ¹w: weak;m: medium; ms: medium-strong; s: strong

TABLE 4C Form C FT-IR Spectroscopy Data Absorption Band¹ FunctionalGroup 1707 s C═O stretch (amide) 1644 ms Acyclic C═N stretch Region1596-1521, C═C, C═N ring stretch and C—N—H (1596 w, 1521 m) bend (amideII/sulphonamide and amine) 1333 ms, 1320 w, SO₂ asymmetric stretch 1313ms 1269 m  909 w  881 s  797 m  703 m  661 m ¹w: weak; m: medium; ms:medium-strong; s: strong

Form A FT-IR

Form A has an FT-IR spectrum that comprises at least one absorption bandselected from the group consisting of 696±2; 1085±2; 1188±2; 1540±2; and3247±3 cm⁻¹. In one embodiment, Form A has an FT-IR spectrum thatcomprises an absorption band at 3247±3 cm'. In another embodiment, FormA has an FT-IR spectrum that comprises an absorption band at 3247±3cm⁻¹, and further comprises at least one absorption band selected fromthe group consisting of 696±2; 1085±2; 1188±2; and 1540±2 cm⁻¹. Inanother embodiment, Form A has an FT-IR spectrum that comprisesabsorption bands at 3247±3 and 696±2 cm⁻¹. In another embodiment, Form Ahas an FT-IR spectrum that comprises absorption bands at 696±2; 1188±2;and 3247±3 cm⁻¹. In another embodiment, Form A has an FT-IR spectrumthat comprises absorption bands at 696±2; 1188±2; 1540±2; and 3247±3cm⁻¹. In another embodiment, Form A has an FT-IR spectrum that comprisesabsorption bands at 696±2; 1085±2; 1188±2; 1540±2; and 3247±3 cm⁻¹.

In another embodiment, Form A has an FT-IR spectrum that (a) comprisesat least one absorption band selected from the group consisting of696±2; 1085±2; 1188±2; 1540±2; and 3247±3 cm⁻¹, and (b) does notcomprise an absorption band at 1645±2 cm⁻¹.

Form B FT-IR

Form B has an FT-IR spectrum that comprises at least one absorption bandselected from the group consisting of 722±2; 920±2; 1211±2; 1395±2; and1452±2 cm⁻¹. In another embodiment, Form B has an FT-IR spectrum thatcomprises an absorption band at 1452±2 cm⁻¹. In another embodiment, FormB has an FT-IR spectrum that comprises an absorption band at 1452±2cm⁻¹, and further comprises at least one additional absorption bandselected from the group consisting of 722±2; 920±2; 1211±2; and 1395±2cm⁻¹. In another embodiment, Form B has an FT-IR spectrum comprisingabsorption bands at 1452±2 and 1395±2 cm⁻¹. In another embodiment, FormB has an IR spectrum comprising absorption bands at 1211±2; 1395±2; and1452±2 cm⁻¹. In another embodiment, Form B has an IR spectrum comprisingabsorption bands at 722±2; 1211±2; 1395±2; and 1452±2 cm⁻¹. In anotherembodiment, Form B has an IR spectrum comprising absorption bands at722±2; 920±2; 1211±2; 1395±2; and 1452±2 cm⁻¹.

In another embodiment, Form B has an FT-IR spectrum that (a) comprisesat least one absorption band selected from the group consisting of722±2; 920±2; 1211±2; 1395±2; and 1452±2 cm⁻¹, and (b) does not comprisean absorption band at 962±2 cm⁻¹.

Form C FT-IR

Form C has an FT-IR spectrum that comprises at least one absorption bandselected from the group consisting of 661±2; 703±2; 797±2; 881±2; 909±2;and 1269±2 cm⁻¹. In another embodiment, Form C has an FT-IR spectrumthat comprises an absorption band at 881±2 cm⁻¹. In another embodiment,Form C has an FT-IR spectrum that comprises an absorption band at 881±2cm⁻¹, and further comprises at least one additional absorption bandselected from the group consisting of 661±2; 703±2; 797±2; 909±2; and1269±2 cm⁻¹. In another embodiment, Form C has an FT-IR spectrumcomprising absorption bands at 881±2 and 661±2 cm⁻¹. In anotherembodiment, Form C has an FT-IR spectrum comprising absorption bands at661±2; 797±2; and 881±2 cm⁻¹. In another embodiment, Form C has an FT-IRspectrum comprising absorption bands at 661±2; 703±2; 797±2; and 881±2cm⁻¹. In another embodiment, Form C has an FT-IR spectrum comprisingabsorption bands at 661±2; 703±2; 797±2; 881±2; and 909±2 cm⁻¹. Inanother embodiment, Form C has an FT-IR spectrum comprising absorptionbands at 661±2; 703±2; 797±2; 881±2; 909±2; and 1269±2 cm⁻¹.

In another embodiment, Form C has an FT-IR spectrum that (a) comprisesat least one absorption band selected from the group consisting of661±2; 703±2; 881±2; 909±2; and 1269±2 cm⁻¹, and (b) does not comprisean absorption band at 688±2 cm⁻¹. In another embodiment, Form C has anFT-IR spectrum that (a) comprises at least one absorption band Selectedfrom the group consisting of 661±2; 703±2; 797±2; 881±2; 909±2; and1269±2 cm⁻¹, and (b) does not comprise an absorption band at 696±2 cm⁻¹.In another embodiment, Form C has an FT-IR spectrum that (a) comprisesat least one absorption band selected from the group consisting of661±2; 703±2; 797±2; 881±2; 909±2; and 1269±2 cm⁻¹, and (b) does notcomprise at least one absorption band selected from the group consistingof 688±2 or 696±2 cm⁻¹.

As previously noted, it is hypothesized that Form A crystallizes asTautomer (1) and Form B and Form C each crystallize as Tautomer (2).FT-IR analysis supports this hypothesis. In particular, the Form C FT-IRspectrum shows a medium-strong absorption band at 1644±2 cm⁻¹ and theForm B FT-IR spectrum shows a strong absorption band at 1646±2 cm⁻¹. Itis believed that these bands correspond to an acyclic C═N stretchingfrequency that is consistent with Tautomer (2). To the contrary, theForm A FT-IR spectrum shows no absorption band at the correspondingfrequency. It is believed that Form A lacks an acyclic C═N stretchingfrequency because it crystallizes as Tautomer (1).

5. Fourier-Transform Raman Spectroscopy

Form A, Form B and Form C were each analyzed using Fourier-TransformRaman (“Raman”) spectroscopy. Raman spectra for Form A, Form B and FormC were obtained using a ThermoNicolet 960 Raman spectrometer. Eachsample (approximately 5 mg) was placed in a glass vial and exposed to1064.5 nm Nd-YAG laser power for excitation. The data were collected at2 cm⁻¹ resolution, measured as Raman intensity as a function of Ramanshift. Data were processed as a Fourier Transform utilizing aHapp-Genzel apodization. Experimental error, unless otherwise noted, was±2 cm⁻¹.

Illustrative Raman spectra for Form A (measurement conditions: 2000scans, laser Power: 750 mW, laser power at the sample: 400 mW), Form B(measurement conditions: 4000 scans, laser power: 600 mW, laser power atthe sample: 340 mW), and Form C (measurement conditions: 960 scans,laser power: 600 mW, laser power at the sample: 340 mW) are shown inFIGS. 11, 12 and 13, respectively. The X-axis is Raman shift (cm⁻¹) andthe Y-axis is intensity. The intensities are intensity assignmentsrelative to the major absorption band in the spectrum and are not basedon absolute values measured from the baseline. Tables 5A, 5B, and 5Clist the corresponding characteristic Raman bands for Form A, Form B andForm C, respectively.

TABLE 5A Form A Raman Spectroscopy Data Band¹ 3255 w² 3040 w 3016 m 2937s 2882 m 1711 s 1608 m 1569 m 1473 w 1418 m 1383 s 1364 m 1335 m 1316 s1285 w 1259 w 1233 m 1165 m  993 m  752 w  701 m  521 m  310 m ¹w: weak;m: medium; s: strong ²Experimental error was ±3 cm⁻¹.

TABLE 5B Form B Raman Spectroscopy Data Band¹ 3054 w 3020 w 2965 w 2936m 2868 m 1706 m 1652 m 1605 s 1535 s 1456 w 1417 m 1376 s 1339 m 1299 s1157 m 1000 m  689 w  536 w  173 w ¹w: weak; m: medium; s: strong

TABLE 5C Form C Raman Spectroscopy Data Band¹ 3084 w 3065 w 3009 m 2988w 2965 m 2930 s 2889 w 1707 s 1651 w 1561 m 1540 m 1447 w 1424 w 1397 w1376 s 1336 s 1316 s 1269 w 1232 w 1161 m 1113 w  999 w  707 w  173 m¹w: weak; m: medium; s: strong

Form A Raman

Form A has a Raman spectrum that comprises at least one band selectedfrom the group consisting of 993±2; 1383±2; 1473±2; 1569±2; and 3255±3cm⁻¹. In another embodiment, Form A has a Raman spectrum that comprisesa band at 3255±3 cm⁻¹. In another embodiment, Form A has a Ramanspectrum that comprises a band at 3255±3 cm⁻¹, and further comprises atleast one additional band selected from the group consisting of 993±2;1383±2; 1473±2; and 1569±2 cm⁻¹. In another embodiment, Form A has aRaman spectrum that comprises bands at 1569±2 and 3255±3 cm⁻¹. Inanother embodiment, Form A has a Raman spectrum that comprises bands at1473±2; 1569±2; and 3255±3 cm⁻¹. In another embodiment, Form A has aRaman spectrum that comprises bands at 1383±2; 1473±2; 1569±2; and3255±3 cm⁻¹. In another embodiment, Form A has a Raman spectrum thatcomprises bands at 993±2; 1383±2; 1473±2; 1569±2; and 3255±3 cm⁻¹.

In another embodiment, Form A has a Raman spectrum that (a) comprises atleast one band selected from the group consisting of 993±2; 1383±2;1473±2; 1569±2; and 3255±3 cm⁻¹, and (b) does not comprise a band at1652±2 cm⁻¹.

Form B Raman

Form B has a Raman spectrum that comprises at least one band selectedfrom the group consisting of 689±2; 1299±2; 1456±2; and 1535±2 cm⁻¹. Inanother embodiment, Form B has a Raman spectrum that comprises a band at1299±2 cm⁻¹. In another embodiment, Form B has a Raman spectrum thatcomprises a band at 1299±2 cm⁻¹, and further comprises at least oneadditional band selected from the group consisting of 689±2; 1456±2; and1535±2 cm⁻¹. In another embodiment, Form B has a Raman spectrumcomprising bands at 689±2 and 1299±2 cm⁻¹. In another embodiment, Form Bhas a Raman spectrum comprising bands at 689±2; 1299±2; and 1535±2 cm⁻¹.In another embodiment, Form B has a Raman spectrum comprising bands at689±2; 1299±2; 1456±2; and 1535±2 cm⁻¹.

In another embodiment, Form B has a Raman spectrum that (a) comprises atleast one band selected from the group consisting of 689±2; 1299±2;1456±2; and 1535±2 cm⁻¹, and (b) does not comprise a band at 1316±2cm⁻¹.

Form C Raman

Form C has a Raman spectrum that comprises at least one band selectedfrom the group consisting of 707±2; 1447±2; and 2988±2 cm⁻¹. In anotherembodiment, Form C has a Raman spectrum that comprises a band at 2988±2cm⁻¹. In another embodiment, Form C has a Raman spectrum with asignificant band at 2988±2 cm⁻¹, and further comprises at least oneadditional band selected from the group consisting of 707±2 and 1447±2cm⁻¹. In another embodiment, Form C has a Raman spectrum comprisingbands at 707±2 and 2988±2 cm⁻¹. In another embodiment, Form C has aRaman spectrum comprising bands at 707±2; 1447±2; and 2988±2 cm⁻¹.

In another embodiment, Form C has a Raman spectrum that (a) comprises atleast one band selected from the group consisting of 707±2; 1447±2; and2988±2 cm⁻¹, and (b) does not comprise a band at 1417±2 cm⁻¹.

As previously noted, it is hypothesized that Form A crystallizes asTautomer (1) and Form B and Form C each crystallize as Tautomer (2).FT-Raman analysis supports this hypothesis. In particular, the Form CFT-Raman spectrum shows a weak Raman band at 1651±2 cm⁻¹ and the Form BFT-Raman spectrum shows a medium Raman band at 1652±2 cm⁻¹. It isbelieved that these bands correspond to an acyclic C═N stretchingfrequency that is consistent with Tautomer (2). To the contrary, theForm A FT-Raman spectrum shows no Raman band at the correspondingfrequency. It is believed that Form A lacks an acyclic C═N stretchingfrequency because it crystallizes as Tautomer (1).

C. Properties of Form A, Form B and Form C

1. Thermodynamic Stability

Form A, Form B and Form C have different thermodynamic stabilities. FormB is more thermodynamically stable than Form A at ambient as well aselevated temperatures (see Example 13, below). Form B and Form C,however, are enantiotropically related. A crossover in the thermodynamicstability of Form B and Form C occurs at a temperature between about 40°C. and about 60° C. (see Example 14, below). In another embodiment, thecrossover in the thermodynamic stability of Form B and Form C occurs ata temperature between about 40° C. and about 50° C. At temperaturesabove this crossover point, Form B is more thermodynamically stable thanForm C. At temperatures below this crossover point (including at ambienttemperatures), Form C is more thermodynamically stable than Form B.

These differences in thermodynamic stability have practical importance.The thermodynamic stability of a crystalline form affects the potentialshelf life of a formulated pharmaceutical product comprising thecrystalline form. Greater thermodynamic stability generally correlateswith longer shelf life for the formulated pharmaceutical product. Inaddition, differences in thermodynamic stability can create issues whereprocessing results in elevated temperatures (e.g., due to milling of thecompound) or processing occurs over a range of temperatures. Suchtemperature changes during processing potentially can result in theconversion of one crystalline form into another crystalline form. If theresulting crystalline form is not the desired form, it may be necessaryto more carefully control the processing temperature(s).

2. Morphology

Form A and Form B also have different crystal morphologies. Althoughfactors such as temperature, solvent, impurities and hydrodynamics(vibrations) can affect crystal morphology, Form A and Form B clearlyhave distinct crystal morphologies. Form A typically exhibits aplate-like morphology. Form B typically exhibits a needle-likemorphology. Form C comprises a mixture of laths, plates and fragmentsthat range in size (maximum dimension) from about 5 microns to about 350microns; typically 50 to 60 microns.

These differences in morphology potentially can affect the ease ofprocessing the compound to prepare a formulated pharmaceutical product.For example, a needle-like morphology can make filtration and processingmore difficult. Alternatively, a plate-like morphology often is moreequi-dimensional resulting in improved flow and handling of the compoundthereby improving the ease of filtration, processing and tableting stepsrelative to a needle-like morphology.

3. Color

Form A, Form B and Form C also have different visual appearances. Form Atypically has a slightly yellowish to ivory coloration. Form B typicallyhas a yellow coloration. Form C typically has a light yellow coloration.The product specification for a formulated pharmaceutical product oftenspecifies not only the chemical purity of the active ingredient, butalso the phase purity of the active ingredient. Batch-to-batchvariability in the crystalline form of an active ingredient generally isnot desirable. In the case ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,the color of a batch can be used for quality control purposes to providea qualitative means of assessing whether the phase purity of that batchsatisfies the desired phase purity standard. In addition, productaesthetics are important and uniformity of color in the finalpharmaceutical product appearance is desirable. Where the colorproperties of a crystalline form affect the appearance of the formulatedproduct, appropriate control of the crystalline form present in theproduct will need to be exercised to maintain color consistency for theproduct.

D. Additional Embodiments

The following are additional embodiments of Form A, Form B and Form C:

Additional Embodiments of Form A

In one embodiment, Form A has a PXRD pattern comprising a diffractionpeak at 8.5±0.1 degrees two theta, and an FT-IR spectrum comprising anabsorption band at 3247±3 cm⁻¹. In another embodiment, Form A has a PXRDpattern comprising a diffraction peak at 8.5±0.1 degrees two theta, anda FT-IR spectrum comprising absorption bands at 3247±3 and 696±2 cm⁻¹.In another embodiment, Form A has a PXRD pattern comprising diffractionpeaks at 8.5±0.1 and 9.0±0.1 degrees two theta, and a FT-IR spectrumcomprising an absorption band at 3247±3 cm⁻¹. In another embodiment,Form A has a PXRD pattern comprising diffraction peaks at 8.5±0.1 and9.0±0.1 degrees two theta, and a FT-IR spectrum comprising absorptionbands at 696±2 cm⁻¹ and 3247±3 cm⁻¹. In another embodiment, Form A has aPXRD pattern comprising diffraction peaks at 8.5±0.1; 9.0±0.1; and16.9±0.1 degrees two theta, and a FT-IR spectrum comprising absorptionbands at 696±2; 1188±2; and 3247±3 cm⁻¹.

In another embodiment, Form A has a PXRD pattern comprising adiffraction peak at 8.5±0.1 degrees two theta, and a Raman spectrumcomprising a band at 3255±3 cm⁻¹. In another embodiment, Form A has aPXRD pattern comprising a diffraction peak at 8.5±0.1 degrees two theta,and a Raman spectrum comprising bands at 1569±2 and 3255±3 cm⁻¹. Inanother embodiment, Form A has a PXRD pattern comprising diffractionpeaks at 8.5±0.1 and 9.0±0.1 degrees two theta, and a Raman spectrumcomprising a band at 3255±3 cm⁻¹. In another embodiment, Form A has aPXRD pattern comprising diffraction peaks at 8.5±0.1 and 9.0±0.1 degreestwo theta, and a Raman spectrum comprising bands at 1569±2 and 3255±3cm⁻¹. In another embodiment, Form A has a PXRD pattern comprisingdiffraction peaks at 8.5±0.1; 9.0±0.1; and 16.9±0.1 degrees two theta,and a Raman spectrum comprising bands at 1569±2 and 3255±3 cm⁻¹. Inanother embodiment, Form A has a PXRD pattern comprising diffractionpeaks at 8.5±0.1; 9.0±0.1; and 16.9±0.1 degrees two theta, and a Ramanspectrum comprising bands at 1473±2; 1569±2; 3255±3 and cm⁻¹.

In another embodiment, Form A has a PXRD pattern comprising adiffraction peak at 8.5±0.1 degrees two theta, and a melting point of174° C.±3° C. In another embodiment, Form A has a PXRD patterncomprising diffraction peaks at 8.5±0.1 and 9.0±0.1 degrees two theta,and a melting point of 174° C.±3° C. In another embodiment, Form A has aPXRD pattern comprising diffraction peaks at 8.5±0.1; 9.0±0.1; and16.9±0.1 degrees two theta, and a melting point of 174° C.±3° C.

In another embodiment, Form A has a PXRD pattern comprising adiffraction peak at 8.5±±0.1 degrees two theta, a FT-IR spectrumcomprising an absorption band at 3247±3 cm⁻¹ and a melting point of 174°C.±3° C. In another embodiment, Form A has a PXRD pattern comprisingdiffraction peaks at 8.5±0.1 and 9.0±0.1 degrees two theta, a FT-IRspectrum comprising an absorption band at 3247±3 cm⁻¹ and a meltingpoint of 174° C.±3° C. In another embodiment, Form A has a PXRD patterncomprising diffraction peaks at 8.5±0.1; 9.0±0.1; and 16.9±0.1 degreestwo theta, a FT-IR spectrum comprising absorption bands at 3247±3 and696±2 cm⁻¹ and a melting point of 174° C.±3° C. In another embodiment,Form A has an a PXRD pattern comprising diffraction peaks at 8.5±0.1;9.0±0.1; and 16.9±0.1 degrees two theta, a FT-IR spectrum comprisingabsorption bands at 696±2; 1188±2; and 3247±3 cm⁻¹, and a melting pointof 174° C.±3° C.

In another embodiment, Form A has a PXRD pattern comprising adiffraction peak at 8.5±0.1 degrees two theta, a FT-IR spectrumcomprising an absorption band at 3247±3 cm⁻¹, and a Raman spectrumcomprising a band at 3255±3 cm⁻¹. In another embodiment, Form A has aPXRD pattern comprising diffraction peaks at 8.5±0.1 and 9.0±0.1 degreestwo theta, a FT-IR spectrum comprising absorption bands at 696±2 and3247±3 cm⁻¹, and a Raman spectrum comprising bands at 1569±2 and 3255±3cm⁻¹. In another embodiment, Form A has a PXRD pattern comprisingdiffraction peaks at 8.5±0.1; 16.9±0.1; and 22.5±0.1, degrees two theta,a FT-IR spectrum comprising absorption bands at 696±2; 1188±2 and 3247±3cm⁻¹, and a Raman spectrum comprising bands at 1569±2 and 3255±3 cm⁻¹.

In another embodiment, Form A has a PXRD pattern comprising diffractionpeaks at 8.5±0.1 and 9.0±0.1 degrees two theta, an FT-IR spectrumcomprising absorption bands at 696±2 and 3247±3 cm⁻¹, an Raman spectrumcomprising bands at 1569±2 and 3255±3 cm⁻¹, and a melting point of 174°C.±3° C. In another embodiment, Form A has a PXRD pattern comprisingdiffraction peaks at; 8.5±0.1; 16.9±0.1; and 22.5±0.1 degrees two theta,an FT-IR spectrum comprising absorption bands at 696±2; 1188±2; and3247±3; cm⁻¹, an Raman spectrum comprising bands at 1569±2 and 3255±3cm⁻¹, and a melting point of 174° C.±3° C.

Additional Embodiments of Form B

In one embodiment, Form B has a PXRD pattern comprising a diffractionpeak at 3.6±0.1 degrees two theta and FT-IR spectrum comprising anabsorption bands at 1452±2 cm⁻¹. In another embodiment, Form B has aPXRD pattern comprising a diffraction peak at 3.6±0.1 degrees two thetaand FT-IR spectrum comprising absorption bands at 1395±2 and 1452±2cm⁻¹. In another embodiment, Form B has a PXRD pattern comprising adiffraction peak at 3.6±0.1 degrees two theta and FT-IR spectrumcomprising absorption bands at 1211±2; 1395±2 and 1452±2 cm⁻¹. Inanother embodiment, Form B has a PXRD pattern comprising a diffractionpeak at 3.6±0.1 degrees two theta and FT-IR spectrum comprisingabsorption bands at 722±2; 920±2; 1211±2; 1395±2 and 1452±2 cm⁻¹. Inanother embodiment, Form B has an a PXRD pattern comprising diffractionpeaks at 3.6±0.1 and 7.2±0.1 degrees two theta and FT-IR spectrumcomprising absorption bands at 1211±2; 1395±2; and 1452±2 cm⁻¹.

In one embodiment, Form B has a PXRD pattern comprising a diffractionpeak at 3.6±0.1 degrees two theta and a melting point of 218° C.±3° C.In another embodiment, Form B has a PXRD pattern comprising diffractionpeaks at 3.6±0.1 and 7.2±0.1 degrees two theta, and a melting point of218° C.±3° C. In another embodiment, Form B has a PXRD patterncomprising diffraction peaks at 3.6±0.1; 7.2±0.1; and 23.8±0.1 degreestwo theta, and a melting point of 218° C.±3° C. In another embodiment,Form B has a PXRD pattern comprising diffraction peaks at 3.6±0.1;7.2±0.1; 10.1±0.1; 14.4±0.1; and 23.8±0.1 degrees two theta, and amelting point of 218° C.±3° C.

In another embodiment, Form B has a PXRD pattern comprising adiffraction peak at 3.6±0.1 degrees two theta, an FT-IR spectrumcomprising an absorption band at 1452±2 cm⁻¹ and a melting point of 218°C.±3° C. In another embodiment, Form B has a PXRD pattern comprising adiffraction peak at 3.6±0.1 degrees two theta, an FT-IR spectrumcomprising absorption bands at 1395±2 cm⁻¹ and 1452±2 cm⁻¹ and a meltingpoint of 218° C.±3° C. In another embodiment, Form B has a PXRD patterncomprising diffraction peaks at 3.6±0.1; 7.2±0.1; 10.1±0.1; 14.4±0.1;and 23.8±0.1 degrees two theta, a FT-IR spectrum comprising absorptionbands at 722±2; 920±2; 1211±2; 1395±2; and 1452±2 cm⁻¹ and a meltingpoint of 218° C.±3° C.

In another embodiment, Form B has a PXRD pattern comprising adiffraction peak at 3.6±0.1 degrees two theta, and a Raman spectrumcomprising an absorption band at 1299±2 cm⁻¹. In another embodiment,Form B has a PXRD pattern comprising a diffraction peak at 3.6±0.1degrees two theta, and a Raman spectrum comprising absorption bands at689±2 and 1299±2 cm⁻¹. In another embodiment, Form B has a PXRD patterncomprising a peak at 3.6±0.1 degrees two theta, and a Raman spectrumcomprising absorption bands at 689±2; 1299±2; and 1535±2 cm⁻¹. Inanother embodiment, Form B has a PXRD pattern comprising a diffractionpeak at 3.6±0.1 degrees two theta, and an Raman spectrum comprisingabsorption bands at 689±2; 1299±2; 1456±2; and 1535±2 cm⁻¹.

In another embodiment, Form B has a PXRD pattern comprising adiffraction peak at 3.6±0.1 degrees two theta, a FT-IR spectrumcomprising an absorption band at 1452±2 cm⁻¹, and a Raman spectrumcomprising an absorption band at 1299±2 cm⁻¹. In another embodiment,Form B has a PXRD pattern comprising a diffraction peak at 3.6±0.1degrees two theta, an FT-IR spectrum comprising absorption bands at1395±2 cm⁻¹ and 1452±2 cm⁻¹, and a Raman spectrum comprising aabsorption bands at 1299±2 and 689±2 cm⁻¹. In another embodiment, Form Bhas a PXRD pattern comprising a diffraction peak at 3.6±0.1 degrees twotheta, a FT-IR spectrum comprising absorption bands at 1395±2 cm⁻¹;1452±2; and 1535±2 cm⁻¹, and a Raman spectrum comprising a absorptionbands at 1299±2 and 689±2 cm⁻¹.

In another embodiment, Form B has a PXRD pattern comprising adiffraction peak at 3.6±0.1 degrees two theta, an FT-IR spectrumcomprising absorption bands at 1395±2 cm⁻¹ and 1452±2 cm⁻¹, a Ramanspectrum comprising an absorption band at 1299±2 cm⁻¹ and a meltingpoint of 218° C.±3° C. In another embodiment, Form B has a PXRD patterncomprising a diffraction peak at 3.6±0.1 degrees two theta, an FT-IRspectrum comprising absorption bands at 1395±2 cm⁻¹; 1452±2; and 1535±2cm⁻¹, a Raman spectrum comprising a absorption bands at 1299±2 and 689±2cm⁻¹ and a melting point of 218° C.±3° C.

Additional Embodiments of Form C

In another embodiment, Form C has a PXRD pattern comprising adiffraction peak at 6.7±0.1 degrees two theta and a FT-IR spectrumcomprising an absorption band at 881±2 cm⁻¹. In another embodiment, FormC has a PXRD pattern comprising a diffraction peak at 6.7±0.1 degreestwo theta and a FT-IR spectrum comprising an absorption band at 881±2cm⁻¹ and 661±2 cm⁻¹. In another embodiment, Form C has a PXRD patterncomprising a diffraction peak at 6.7±0.1 degrees two theta and a FT-IRspectrum comprising an absorption band at 881±2; 797±2; 703±2; and 661±2cm⁻¹. In another embodiment, Form C has a PXRD pattern comprising adiffraction peak at 6.7±0.1 and 26.1±0.1 degrees two theta and a FT-IRspectrum comprising an absorption band at 881±2 cm⁻¹ and 661±2 cm⁻¹.

In another embodiment, Form C has a PXRD pattern comprising adiffraction peak at 6.7±0.1 degrees two theta and a melting point of188° C.±3° C. In another embodiment, Form C has a PXRD patterncomprising diffraction peaks at 6.7±0.1 and 26.1±0.1 degrees two thetaand a melting point of 188° C.±3° C. In another embodiment, Form C has aPXRD pattern comprising diffraction peaks at 6.7±0.1; 20.2±0.1; and17.7±0.1 degrees two theta and a melting point of 188° C.±3° C. Inanother embodiment, Form C has a PXRD pattern comprising diffractionpeaks at 6.7±0.1; 10.6±0.1; 14.0±0.1; 17.7±0.1; and 20.2±0.1 degrees twotheta and a melting point of 188° C.±3° C.

In another embodiment, Form C has a PXRD pattern comprising adiffraction peak at 6.7±0.1 degrees two theta and a Raman spectrumcomprising an absorption band at 2988±2 cm⁻¹. In another embodiment,Form C has a PXRD pattern comprising a diffraction peak at 6.7±0.1degrees two theta and a Raman spectrum comprising absorption bands at707±2 and 2988±2 cm⁻¹. In another embodiment, Form C has a PXRD patterncomprising a diffraction peak at 6.7±0.1 degrees two theta and a Ramanspectrum comprising absorption bands at 707±2; 1447±2; and 2988±2 cm⁻¹.In another embodiment, Form C has a PXRD pattern comprising diffractionpeaks at 6.7±0.1 and 26.1±0.1 degrees two theta and a Raman spectrumcomprising absorption bands at 707±2; 1447±2; and 2988±2 cm⁻¹.

In another embodiment, Form C has a PXRD pattern comprising adiffraction peak at 6.7±0.1 degrees two theta, a FT-IR spectrumcomprising absorption bands at 661±2 cm⁻¹ and 881±2 cm⁻¹ and a meltingpoint of 188° C.±3° C. In another embodiment, Form C has a PXRD patterncomprising diffraction peaks at 6.7±0.1 and 20.2±0.1 degrees two theta,a FT-IR spectrum comprising absorption bands at 661±2, 881±2 and 797±2cm⁻¹ and a melting point of 188° C.±3° C.

In another embodiment, Form C has a PXRD pattern comprising adiffraction peak at 6.7±0.1 degrees two theta, a FT-IR spectrumcomprising an absorption band at 881±2 cm⁻¹, an Raman spectrumcomprising an absorption band at 2988±2 cm⁻¹ and a melting point of 188°C.±3° C. In another embodiment, Form C has a PXRD pattern comprising adiffraction peak at 6.7±0.1 degrees two theta, a FT-IR spectrumcomprising absorption bands at 661±2 cm⁻¹ and 881±2 cm⁻¹, an Ramanspectrum comprising an absorption band at 2988±2 cm⁻¹ and a meltingpoint of 188° C.±3° C. In another embodiment, Form C has a PXRD patterncomprising diffraction peaks at 6.7±0.1 and 20.2±0.1 degrees two theta,a FT-IR spectrum comprising absorption bands at 661±2 cm⁻¹ and 881±2cm⁻¹, an Raman spectrum comprising absorption bands at 707±2 and 2988±2cm⁻¹ and a melting point of 188° C.±3° C. In another embodiment, Form Chas a PXRD pattern comprising diffraction peaks at 6.7±0.1 and 20.2±0.1degrees two theta, a FT-IR spectrum comprising absorption bands at881±2; 797±1; 703±2; and 661±2 cm⁻¹, an Raman spectrum comprisingabsorption bands at 707-±˜2; 1447±2 and 2988±2 cm⁻¹ and a melting pointof 188° C.±3° C.

E. Phase Pure Forms and Combinations of Form A, Form B, and Form C

Each of Form A, Form B, and Form C can be obtained as a substantiallyphase pure form. Alternatively, each of Form A, Form B, and Form C canbe present in combination with one or more of the other forms.

In one embodiment, the invention comprisesN[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 50% by weight of the compound is Form A. Inadditional embodiments, the invention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 20%, at least about 30%, or at least about 40% byweight of the compound is Form A. In additional embodiments, theinvention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 60%, at least about 70%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99% byweight of the compound is Form A. In another embodiment, the inventioncomprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidethat is substantially phase pure Form A.

In one embodiment, the invention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 50% by weight of the compound is Form B. Inadditional embodiments, the invention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 20%, at least about 30%, or at least about 40% byweight of the compound is Form B. In additional embodiments, theinvention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 60%, at least about 70%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99% byweight of the compound is Form B. In another embodiment, the inventioncomprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidethat is substantially phase pure Form B.

In one embodiment, the invention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 50% by weight of the compound is Form C. Inadditional embodiments, the invention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 20%, at least about 30%, or at least about 40% byweight of the compound is Form C. In additional embodiments, theinvention comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide,wherein at least about 60%, at least about 70%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99% byweight of the compound is Form C. In another embodiment, the inventioncomprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidethat is substantially phase pure Form C.

F. Methods For Preparing Form A, Form B, and Form C

The present invention also comprises methods for preparing Form A, FormB, and Form C. Representative methods are disclosed in the examplescontained in this application.

The invention further comprises each of Form A, Form B, and Form Cprepared in accordance with the methods disclosed in this application.In one embodiment, the invention comprises Form A prepared in accordancewith such methods. In another embodiment, the invention comprises Form Bprepared in accordance with such methods. In another embodiment theinvention comprises Form C prepared in accordance with such methods.

G. Pharmaceutical Compositions

Form A, Form B, and Form C and combinations of such forms can beadministered by any suitable route, preferably in the form of apharmaceutical composition adapted to such a route, and in a doseeffective for the treatment intended. Accordingly, the inventionspecifically comprises pharmaceutical compositions comprising at leastone anhydrous crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidein association with one or more pharmaceutically-acceptable carriers.The amount of Form A, Form B, and/or Form C that is administered and thedosage regimen for treating a condition or disorder with Form A, Form B,and/or Form C depends on a variety of factors, including the age,weight, sex and medical condition of the subject, the severity of thedisease, the route and frequency of administration, and the particularcompound employed, and thus may vary widely. The pharmaceuticalcompositions may contain Form A, Form B, and/or Form C in the range ofabout 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg andmost preferably between about 1 and 200 mg. A daily dose of about 0.01to 100 mg/kg body weight, preferably between about 0.5 and about 20mg/kg body weight and most preferably between about 0.1 to 10 mg/kg bodyweight, may be appropriate. The daily dose can be administered in one tofour doses per day.

In one embodiment, the pharmaceutical composition comprises Form A and apharmaceutically-acceptable carrier. In another embodiment, thepharmaceutical composition comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidethat is substantially phase pure Form A, and apharmaceutically-acceptable carrier. In another embodiment, thepharmaceutical composition comprises Form B and apharmaceutically-acceptable carrier. In another embodiment, thepharmaceutical composition comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidethat is substantially phase pure Form B, and apharmaceutically-acceptable carrier. In another embodiment, thepharmaceutical composition comprises Form C and apharmaceutically-acceptable carrier. In another embodiment, thepharmaceutical composition comprisesN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidethat is substantially phase pure Form C, and apharmaceutically-acceptable carrier.

In yet another embodiment, the pharmaceutical composition comprises acombination of at least two forms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideselected from the group consisting of Form A, Form B, and Form C and apharmaceutically-acceptable carrier. In one embodiment, the weight ratioof the amount of the first form to the second form is at least about1:1. In other embodiments, this ratio is at least about 3:2; at leastabout 7:3; at least about 4:1; at least about 9:1; at least about 95:5;at least about 96:4; at least about 97:3; at least about 98:2; or atleast about 99:1. In another embodiment, the pharmaceutical compositioncomprises three forms ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideselected from the group consisting of Form A, Form B, and Form C and apharmaceutically-acceptable carrier.

H. Methods of Treatment

The present invention further comprises methods for treating a conditionin a subject having or susceptible to having such a condition, byadministering to the subject a therapeutically-effective amount of oneor more compounds of Form A, Form B, Form C or combinations of suchforms as described above. In one embodiment, the treatment ispreventative treatment. In another embodiment, the treatment ispalliative treatment. In another embodiment, the treatment isrestorative treatment.

The conditions that can be treated in accordance with the presentinvention are PDE-5 mediated conditions. Such conditions includecardiovascular diseases, metabolic diseases, central nervous systemdiseases, pulmonary diseases, sexual dysfunction, and renal dysfunction.

In one embodiment, the condition is a cardiovascular disease,particularly a cardiovascular disease selected from the group consistingof hypertension (such as essential hypertension, pulmonary hypertension,secondary hypertension, isolated systolic hypertension, hypertensionassociated with diabetes, hypertension associated with atherosclerosis,and renovascular hypertension); complications associated withhypertension (such as vascular organ damage, congestive heart failure,angina, stroke, glaucoma and impaired renal function); valvularinsufficiency; stable, unstable and variant (Prinzmetal) angina;peripheral vascular disease; myocardial infarct; stroke; thromboembolicdisease; restenosis; arteriosclerosis; atherosclerosis; pulmonaryarterial hypertension; angiostenosis after bypass; angioplasty (such aspercutaneous transluminal angioplasty, or percutaneous transluminalcoronary angioplasty); hyperlipidemia; hypoxic vasoconstriction;vasculitis, such as Kawasaki's syndrome; heart failure (such ascongestive, decompensated, systolic, diastolic and left ventricularheart failure; right ventricular heart failure; and left ventricularhypertrophy); Raynaud's disease; preeclampsia; pregnancy-induced highblood pressure; cardiomyopathy; and arterial occlusive disorders.

In another embodiment, the condition is hypertension. In anotherembodiment, the condition is pulmonary arterial hypertension. In anotherembodiment, the condition is heart failure. In another embodiment, thecondition is diastolic heart failure. In another embodiment, thecondition is systolic heart failure. In another embodiment, thecondition is angina. In another embodiment, the condition is thrombosis.In another embodiment, the condition is stroke.

In another embodiment, the condition is a metabolic disease,particularly a metabolic disease selected from the group consisting ofSyndrome X; insulin resistance or impaired glucose tolerance; diabetes(such as type I and type II diabetes); syndromes of insulin resistance(such as insulin receptor disorders, Rabson-Mendenhall syndrome,leprechaunism, Kobberling-Dunnigan syndrome, Seip syndrome, Lawrencesyndrome, Cushing syndrome, acromegaly, pheochomocytoma, glucagonoma,primary aldosteronism, somatostatinoma, Lipoatrophic diabetes, β-celltoxin induced diabetes, Grave's disease, Hashimoto's thyroiditis andidiopathic Addison's disease); diabetic complications (such as diabeticgangrene, diabetic arthropathy, diabetic nephropathy, diabeticglomerulosclerosis, diabetic deramatopathy, diabetic neuropathy,peripheral diabetic neuropathy, diabetic cataract, and diabeticretinopathy); hyperglycemia; and obesity.

In another embodiment, the condition is insulin resistance. In anotherembodiment, the condition is nephropathy.

In another embodiment, the condition is a disease of the central nervoussystem, particularly a disease of the central nervous system selectedfrom the group consisting of vascular dementia; craniocerebral trauma;cerebral infarcts; dementia; concentration disorders; Alzheimer'sdisease; Parkinson's disease; amyolateral sclerosis (ALS); Huntington'sdisease; multiple sclerosis; Creutzfeld-Jacob; anxiety; depression;sleep disorders; and migraine. In one embodiment, the condition isAlzheimer's disease. In another embodiment, the condition is Parkinson'sdisease. In one embodiment, the condition is ALS. In another embodiment,the condition is a concentration disorder.

In one embodiment, the condition is a pulmonary disease, particularly apulmonary disease selected from the group consisting of asthma; acuterespiratory distress; cystic fibrosis; chronic obstructive pulmonarydisease (COPD); bronchitis; and chronic reversible pulmonaryobstruction.

In one embodiment, the condition is sexual dysfunction, particularlysexual dysfunction selected from the group consisting of impotence(organic or psychic); male erectile dysfunction; clitoral dysfunction;sexual dysfunction after spinal cord injury; female sexual arousaldisorder; female sexual orgasmic dysfunction; female sexual paindisorder; and female hypoactive sexual desire disorder. In anotherembodiment, the condition is erectile dysfunction.

In another embodiment, the condition is renal dysfunction, particularlya renal dysfunction selected from the group consisting of acute orchronic renal failure; nephropathy (such as diabetic nephropathy);glomerulopathy; and nephritis.

In another embodiment, the condition is pain. In another embodiment, thecondition is acute pain. Examples of acute pain include acute painassociated with injury or surgery. In another embodiment, the conditionis chronic pain. Examples of chronic pain include neuropathic pain(including postherpetic neuralgia and pain associated with peripheral,cancer or diabetic neuropathy), carpal tunnel syndrome, back pain(including pain associated with herniated or ruptured intervertabraldiscs or abnormalities of the lumber facet joints, sacroiliac joints,paraspinal muscles or the posterior longitudinal ligament), headache,cancer pain (including tumour related pain such as bone pain, headache,facial pain or visceral pain) or pain associated with cancer therapy(including postchemotherapy syndrome, chronic postsurgical painsyndrome, post radiation syndrome, pain associated with immunotherapy,or pain associated with hormonal therapy), arthritic pain (includingosteoarthritis and rheumatoid arthritis pain), chronic post-surgicalpain, post herpetic neuralgia, trigeminal neuralgia, HIV neuropathy,phantom limb pain, central post-stroke pain and pain associated withchronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinalcord injury, Parkinson's disease, epilepsy and vitamin deficiency. Inanother embodiment, the condition is nociceptive pain (including painfrom central nervous system trauma, strains/sprains, burns, myocardialinfarction and acute pancreatitis, post-operative pain (pain followingany type of surgical procedure), posttraumatic pain, renal colic, cancerpain and back pain). In another embodiment, the condition is painassociated with inflammation (including arthritic pain (such asosteoarthritis and rheumatoid disease pain), ankylosing spondylitis,visceral pain (including inflammatory bowel disease, functional boweldisorder, gastro-esophageal reflux, dyspepsia, irritable bowel syndrome,functional abdominal pain syndrome, Crohn's disease, ileitis, ulcerativecolitis, dysmenorrheal, cystitis, pancreatitis and pelvic pain). Inanother embodiment, the condition is pain resulting frommusculo-skeletal disorders (including myalgia, fibromyalgia,spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articularrheumatism, dystrophinopathy, glycogenolysis, polymyositis andpyomyositis). In another embodiment, the condition is selected from thegroup consisting of heart and vascular pain (including pain caused byangina, myocardical infarction, mitral stenosis, pericarditis, Raynaud'sphenomenon, scleredoma and skeletal muscle ischemia). In anotherembodiment, the condition is selected from the group consisting of headpain (including migraine such as migraine with aura and migraine withoutaura), cluster headache, tension-type headache mixed headache andheadache associated with vascular disorders; orofacial pain, includingdental pain, otic pain, burning mouth syndrome and temporomandibularmyofascial pain).

In another embodiment, the condition is a urologic condition selectedfrom the group consisting of bladder outlet obstruction; incontinenceand benign prostatic hyperplasia.

In another embodiment, the condition is an ophthalmic condition selectedfrom retinal disease; macular degeneration and glaucoma.

In another embodiment, the condition is selected from the groupconsisting of tubulointerstitial disorders; anal fissure; baldness;cancerous cachexia; cerebral apoplexy; disorders of gut motility;enteromotility disorders; dysmenorrhoea (primary and secondary);glaucoma; macular degeneration; antiplatelet; haemorrhoids;incontinence; irritable bowel syndrome (IBS); tumor metastasis; multiplesclerosis; neoplasia; nitrate intolerance; nutcracker oesophagus;osteoporosis; infertility; premature labor; psoriasis; retinal disease;skin necrosis; and urticaria. In another embodiment, the condition isosteoporosis.

In another embodiment, the condition is associated with endothelialdysfunction, particularly conditions selected from the group consistingof atherosclerotic lesions, myocardial ischaemia, peripheral ischaemia,valvular insufficiency, pulmonary arterial hypertension, angina,vascular complications after vascular bypass, vascular dilation,vascular repermeabilisation, and heart transplantation.

The methods and compositions of the present invention are suitable foruse with, for example, mammalian subjects such as humans, other primates(e.g., monkeys, chimpanzees), companion animals (e.g., dogs, cats,horses), farm animals (e.g., goats, sheep, pigs, cattle), laboratoryanimals (e.g., mice, rats), and wild and zoo animals (e.g., wolves,bears, deer). In another embodiment, the subject is a human.

I. Use in the Preparation of a Medicament

The present invention further comprises methods for the preparation of apharmaceutical composition (or “medicament) comprising Form A, Form B,Form C or combinations of such forms, in combination with one or morepharmaceutically-acceptable carriers and/or other active ingredients foruse in treating the conditions described above.

J. Working Examples Example 1 Preparation ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide

N-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

Step 1 Dimethyl 1-(2-ethoxyethyl)-4-nitro-1H-pyrazole-3,5-dicarboxylate

Dimethyl 4-nitro-1H-pyrazole-3,5-dicarboxylate (2.0 g, 8.83 mmol,WO00/24745, page 48, preparation 2) was added to a solution of2-ethoxyethyl bromide (1.18 mL, 10.45 mmol) and potassium carbonate(1.32 g, 9.56 mmol) in N,N-dimethylformamide (35 mL) and the reactionmixture stirred for 48 hours at room temperature. The reaction mixturewas concentrated in vacuo and the residue partitioned between ethylacetate (200 mL) and water (100 mL). The organic layer was separated,dried over magnesium sulphate and concentrated in vacuo. The crudeproduct was purified by column chromatography on silica gel eluting withpentane:ethyl acetate 100:0 to 70:30 to yield the title product, 1.63 g.

¹H NMR (CDCl₃, 400 MHz) δ: 1.07 (s, 3H), 3.41 (q, 2H), 3.73 (t, 2H),3.89 (s, 3H), 3.94 (s, 3H), 4.76 (t, 2H). MS APCI+m/z 302, [MH]⁺.

Step 2 4-Nitro-1-(2-ethoxyethyl)-1H-pyrazole-3,5-dicarboxylic acid3-methyl ester

The ester of Step 1 (1.63 g, 5.4 mmol) was added to a solution ofpotassium hydroxide (330 mg, 5.9 mmol) in methanol (20 mL) and thereaction mixture stirred at room temperature for 18 hours. The reactionmixture was concentrated in vacuo and the crude product dissolved inwater and washed with ether. The aqueous phase was acidified with 2Mhydrochloric acid and extracted into dichloromethane (3×100 mL). Theorganics were combined, dried over magnesium sulphate and concentratedin vacuo to yield the title product.

¹H NMR (CD₃OD, 400 MHz) δ: 1.07 (s, 3H), 3.47 (q, 2H), 3.80 (t, 2H),3.88 (s, 3H), 4.77 (t, 2H). MS APCI+m/z 288 [MH]⁺.

Step 3 Methyl5-carbamoyl-1-(2-ethoxyethyl)-4-nitro-1H-pyrazole-3-carboxylate

Oxalyl chloride (1.2 mL, 13.76 mmol) and N,N-dimethylformamide (39 μL)were added to a solution of the carboxylic acid of Step 2 (1.33 g, 4.63mmol) in dichloromethane (20 mL) and the reaction mixture stirred atroom temperature for 2 hours. The reaction mixture was concentrated invacuo and azeotroped from dichloromethane (3×50 mL). The product wasdissolved in tetrahydrofuran (50 mL), cooled in an ice bath, treatedwith 0.88 ammonia solution (10 mL) and stirred for 18 hours at roomtemperature. The mixture was concentrated in vacuo and the residuepartitioned between dichloromethane (200 mL) and water (50 mL). Theorganics phase was dried over magnesium sulphate and concentrated invacuo to yield the title product.

¹H NMR (DMSO-D₆, 400 MHz) δ: 1.06 (t, 3H), 3.40 (m, 2H), 3.77 (m, 2H),3.84 (s, 3H), 4.38 (m, 2H), 8.35 (m, 1H), 8.46 (m, 1H). MS APCI+m/z 287[MH]⁺.

Step 4 Methyl4-amino-5-carbamoyl-1-(2-ethoxyethyl)-1H-pyrazole-3-carboxylate

Palladium(II) hydroxide (100 mg) was added to a solution of the nitrocompound of Step 3 (970 mg, 3.39 mmol) in methanol (20 mL) and themixture warmed to reflux. Ammonium formate (1.07 g, 16.97 mmol) wasadded and the reaction mixture stirred at reflux for 2 hours. Thecatalyst was removed by filtration through Arbocel® and the reactionmixture concentrated in vacuo to yield the title product.

¹H NMR (DMSO-D₆, 400 MHz) δ: 1.02 (t, 3H), 3.33 (m, 2H), 3.66 (m, 2H),3.80 (s, 3H), 4.57 (m, 2H), 5.11 (m, 2H), 7.49 (m, 2H), MS APCI+m/z 257[MH]⁺.

Step 5 Methyl1-(2-ethoxyethyl)-5,7-dioxo-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate

A solution of the amide of Step 4 (570 mg, 3.38 mmol) inN,N-dimethylformamide (30 mL) was treated with N,N′-carbonyldiimidazole(658 mg, 4.06 mmol) and the reaction mixture stirred at room temperaturefor 1 hour and then at 90° C. for 18 hours. The reaction mixture wasconcentrated in vacuo and the crude product suspended in acetone andsonicated for 30 minutes. The solid product was filtered off and driedin vacuo to yield the title product.

¹H NMR (DMSO-D₆, 400 MHz) δ: 1.02 (t, 3H), 3.37 (m, 2H), 3.77 (m, 2H),3.83 (s, 3H), 4.63 (m, 2H), 10.75 (s, 1H), 11.40 (s, 1H). MS ES−m/z 281[M-H]⁻.

Step 6 Methyl5,7-dichloro-1-(2-ethoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate

Phosphorous oxychloride (934 μL, 10.0 mmol) and tetraethylammoniumchloride (195 mg, 1.50 mmol) were added to a solution of the dione ofStep 5 (140 mg, 0.50 mmol) in propionitrile (5 mL) and the reactionmixture refluxed for 18 hours. The reaction mixture was concentrated invacuo and the crude product partitioned between ethyl acetate (50 mL)and water (50 mL). The organic layer was dried over magnesium sulphateand concentrated in vacuo. The crude product was purified by columnchromatography on silica gel eluting with pentane:ethyl acetate 100:0 to75:25 to yield the title product.

¹H NMR (CDCl₃, 400 MHz) δ: 1.05 (t, 3H), 3.41 (m, 2H), 3.84 (m, 2H),4.06 (s, 3H), 5.00 (m, 2H). MS APCI+m/z 319 [MH]⁺.

Step 7 Methyl5-chloro-1-(2-ethoxyethyl)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate

The dichloro compound of Step 6 (1.98 g, 6.20 mmol) was dissolved indimethyl sulphoxide (10 mL) and the solution treated with2-amino-4-methylpyridine (1.34 g, 12.4 mmol). The reaction mixture wasstirred at room temperature for 18 hours. The reaction mixture waspartitioned between dichloromethane (300 mL) and water (500 mL) and thedichloromethane layer separated. The organic phase was washed with water(3×100 mL), dried over magnesium sulphate and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel eluting withdichloromethane:methanol 100:0 to 98:2. The crude product was trituratedwith ether (50 mL), filtered and concentrated in vacuo to yield thetitle product, 1.2 g.

¹H-NMR (CDCl₃, 400 MHz) δ: 1.06 (t, 3H), 2.49 (s, 3H), 3.62 (m, 2H),4.00 (t, 2H), 4.06 (s, 3H), 5.05 (m, 2H), 6.98 (m, 1H), 8.16 (m, 1H),8.50 (m, 1H). MS APCI+m/z 391 [MH]⁺.

Step 85-Chloro-7-(4-methylpyridin-2-yl-amino)-1-(2-ethoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylicacid

The ester of Step 7 (4.3 g, 11 mmol) was dissolved in dioxan (50 mL) andthe solution treated with a 1M aqueous solution of sodium hydroxide(22.0 mL, 22.0 mmol). The reaction mixture was then stirred for 18 hoursat room temperature. The reaction mixture was evaporated to dryness, theresidue dissolved in water (100 mL) and washed with dichloromethane (50mL). The aqueous phase was then acidified with 1M citric acid solutionto pH 4-5 and a yellow precipitate formed. The mixture was stirred for15 minutes before being filtered and the solid product dried in vacuoover phosphorus pentoxide to yield the title product, 3.75 g.

¹H NMR (DMSO-D₆, 400 MHz) δ: 1.00 (t, 3H), 2.34 (s, 3H), 3.45 (m, 2H),3.81 (m, 2H), 4.84 (m, 2H), 6.93 (m, 1H), 7.89 (m, 1H), 8.16 (m, 1H).

Step 9N-[5-Chloro-1-(2-ethoxyethyl)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide

The carboxylic acid of Step 8 (1.0 g, 2.70 mmol), methanesulphonamide(330 mg, 3.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (660 mg, 3.5 mmol) and 4-dimethylaminopyridine (390 mg,3.5 mmol) were dissolved in N,N-dimethylformamide (10 mL) and thereaction mixture stirred at room temperature for 60 hours. Additionalmethanesulphonamide (165 mg, 1.7 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (330 mg, 1.7mmol) and 4-dimethylaminopyridine (195 mg, 1.7 mmol) were added and thereaction mixture stirred for a further 20 hours. Furthermethanesulphonamide (165 1.7 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (330 mg, 1.7mmol) and 4-dimethylaminopyridine (195 mg 1.7 mmol) were added and thereaction mixture stirred for a final 18 hours. The reaction mixture wasconcentrated in vacuo and the residue partitioned betweendichloromethane (25 mL) and water (25 mL). The organic phase wasseparated, washed with water (2×25 mL), dried over magnesium sulphateand concentrated in vacuo. The residue was purified by columnchromatography on silica gel eluting withdichloromethane:methanol:acetic acid 100:0:0 to 96:3.5:0.5. The crudeproduct was triturated in warm ethyl acetate (10 mL) to yield the titleproduct, 290 mg.

¹H NMR (DMSO-D₆, 400 MHz) δ: 0.95 (t, 3H), 2.40 (s, 3H), 3.40 (s, 3H),3.45 (d, 2H), 3.85 (m, 2H), 4.95 (m, 2H), 7.15 (d, 1H), 7.85 (s, 1H),8.25 (d, 1H). MS ES−m/z 452 [M-H]⁻.

Step 10N-[1-(2-Ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide

The chloro compound of Step 9 (110 mg, 0.24 mmol), NI-methyl-ethylamine(79 mg, 1.2 mmol), N-ethyldiisopropylamine (210 μL, 1.20 mmol) andcaesium fluoride (37 mg, 0.24 mmol) were dissolved in dimethylsulphoxide (1 mL) and the reaction mixture heated to 110° C. for 5 hoursin a ReactiVial™. The reaction mixture was partitioned between ethylacetate (10 mL) and water (10 mL) and the organic phase separated andwashed with water (2×10 mL). The organic phase was then dried overmagnesium sulphate and concentrated in vacuo. The residue was purifiedby column chromatography on silica gel eluting withdichloromethane:methanol 99:1 to 97:3. The purified material wasevaporated and dried to yield a pale yellow solid (66 mg). The PXRDpattern for the solid is shown as FIG. 14.

¹H NMR (DMSO-D₆+CF₃CO₂D, 400 MHz) δ: 0.99 (t, 3H), 1.17 (t, 3H), 2.44(s, 3H), 3.18 (s, 3H), 3.41 (s, 3H), 3.44 (d, 2H), 3.66 (d, 2H), 3.88(t, 2H), 4.93 (t, 2H), 7.16 (d, 1H), 8.09 (s, 1H), 8.26 (d, 1H). MSES−m/z 475 [M-H]⁻.

Example 2 Preparation ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide

An alternative synthetic scheme for the preparation ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideis described in the synthetic scheme shown as FIG. 15.

Example 3 Preparation of Form A (Recrystallization From Ethyl Acetate)

Form A crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

Step 1 Dimethyl 1-(2-ethoxyethyl)-4-nitro-1H-pyrazole-3,5-dicarboxylate

Dimethyl 4-nitro-1H-pyrazole-3,5-dicarboxylate (WO00/24745, page 48,preparation 2) (2.0 g, 8.83 mmol) was added to a solution of2-ethoxyethyl bromide (1.18 mL, 10.45 mmol) and potassium carbonate(1.32 g, 9.56 mmol) in N,N-dimethylformamide (35 mL) and the reactionmixture stirred for 48 hours at room temperature. The reaction mixturewas concentrated in vacuo and the residue partitioned between ethylacetate (200 mL) and water (100 mL). The organic layer was separated,dried over magnesium sulphate and concentrated in vacuo. The crudeproduct was purified by column chromatography on silica gel eluting withpentane: ethyl acetate 100:0 to 70:30 to yield the ester product, 1.63g.

¹H NMR (CDCl₃, 400 MHz) δ: 1.07 (s, 3H), 3.41 (q, 2H), 3.73 (t, 2H),3.89 (s, 3H), 3.94 (s, 3H), 4.76 (t, 2H). MS APCI+m/z 302, [MH]⁺.

Step 2 4-Nitro-1-(2-ethoxyethyl)-1H-pyrazole-3,5-dicarboxylic acid3-methyl ester

The ester of step 1 (1.63 g, 5.4 mmol) was added to a solution ofpotassium hydroxide (330 mg, 5.9 mmol) in methanol (20 mL) and thereaction mixture stirred at room temperature for 18 hours. The reactionmixture was concentrated in vacuo and the crude product dissolved inwater and washed with ether. The aqueous phase was acidified with 2Mhydrochloric acid and extracted into dichloromethane (3×100 mL). Theorganics were combined, dried over magnesium sulphate and concentratedin vacuo to yield the nitro product.

¹H NMR (CD₃OD, 400 MHz) δ: 1.07 (s, 3H), 3.47 (q, 2H), 3.80 (t, 2H),3.88 (s, 3H), 4.77 (t, 2H). MS APCI+m/z 288 [MH]⁺.

Step 3 Methyl5-carbamoyl-1-(2-ethoxyethyl)-4-nitro-1H-pyrazole-3-carboxylate

Oxalyl chloride (1.2 mL, 13.76 mmol) and N,N-dimethylformamide (39 μL)were added to a solution of the carboxylic acid of step 2 (1.33 g, 4.63mmol) in dichloromethane (20 mL) and the reaction mixture stirred atroom temperature for 2 hours. The reaction mixture was concentrated invacuo and azeotroped from dichloromethane (3×50 mL). The product wasdissolved in tetrahydrofuran (50 mL), cooled in an ice bath, treatedwith 0.88 ammonia solution (10 mL) and stirred for 18 hours at roomtemperature. The mixture was concentrated in vacuo and the residuepartitioned between dichloromethane (200 mL) and water (50 mL). Theorganics phase was dried over magnesium sulphate and concentrated invacuo to yield the nitro product. ¹H NMR (DMSO-D₆, 400 MHz) δ: 1.06 (t,3H), 3.40 (m, 2H), 3.77 (m, 2H), 3.84 (s, 3H), 4.38 (m, 2H), 8.35 (m,1H), 8.46 (m, 1H). MS APCI+m/z 287 [MH]⁺.

Step 4 Methyl4-amino-5-carbamoyl-1-(2-ethoxyethyl)-1H-pyrazole-3-carboxylate

Palladium (II) hydroxide (100 mg) was added to a solution of the nitrocompound of step 3 (970 mg, 3.39 mmol) in methanol (20 mL) and themixture warmed to reflux. Ammonium formate (1.07 g, 16.97 mmol) wasadded and the reaction mixture stirred at reflux for 2 hours. Thecatalyst was removed by filtration through Arbocel® and the reactionmixture concentrated in vacuo to yield the amide product.

¹H NMR (DMSO-D₆, 400 MHz) δ: 1.02 (t, 3H), 3.33 (m, 2H), 3.66 (m, 2H),3.80 (s, 3H), 4.57 (m, 2H), 5.11 (m, 2H), 7.49 (m, 2H). MS APCI+m/z 257[MH]⁺.

Step 5 Methyl1-(2-ethoxyethyl)-5,7-dioxo-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate

A solution of the amide of step 4 (570 mg, 3.38 mmol) inN,N-dimethylformamide (30 mL) was treated with N,N′-carbonyldiimidazole(658 mg, 4.06 mmol) and the reaction mixture stirred at room temperaturefor 1 hour and then at 90° C. for 18 hours. The reaction mixture wasconcentrated in vacuo and the crude product suspended in acetone andsonicated for 30 minutes. The solid product was filtered off and driedin vacuo to yield the dione product. ¹H NMR (DMSO-D₆, 400 MHz) δ: 1.02(t, 3H), 3.37 (m, 2H), 3.77 (m, 2H), 3.83 (s, 3H), 4.63 (m, 2H), 10.75(s, 1H), 11.40 (s, 1H). MS ES−m/z 281 [M-H]⁻.

Step 6 Methyl5,7-dichloro-1-(2-ethoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate

Phosphorous oxychloride (934 μL, 10.0 mmol) and tetraethylammoniumchloride (195 mg, 1.50 mmol) were added to a solution of the dione ofstep 5 (140 mg, 0.50 mmol) in propionitrile (5 mL) and the reactionmixture refluxed for 18 hours. The reaction mixture was concentrated invacuo and the crude product partitioned between ethyl acetate (50 mL)and water (50 mL). The organic layer was dried over magnesium sulphateand concentrated in vacuo. The crude product was purified by columnchromatography on silica gel eluting with pentane:ethyl acetate 100:0 to75:25 to yield the dichloro product.

¹H NMR (CDCl₃, 400 MHz) δ: 1.05 (t, 3H), 3.41 (m, 2H), 3.84 (m, 2H),4.06 (s, 3H), 5.00 (m, 2H). MS APCI+m/z 319 [MH]⁺.

Step 7 Methyl5-chloro-1-(2-ethoxyethyl)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate

The dichloro compound of step 6 (1.98 g, 6.20 mmol) was dissolved indimethyl sulphoxide (10 mL) and the solution treated with2-amino-4-methylpyridine (1.34 g, 12.4 mmol). The reaction mixture wasstirred at room temperature for 18 hours. The reaction mixture waspartitioned between dichloromethane (300 mL) and water (500 mL) and thedichloromethane layer separated. The organic phase was washed with water(3×100 mL), dried over magnesium sulphate and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel eluting withdichloromethane:methanol 100:0 to 98:2. The crude product was trituratedwith ether (50 mL), filtered and concentrated in vacuo to yield themonochloro product, 1.2 g. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.06 (t, 3H), 2.49(s, 3H), 3.62 (m, 2H), 4.00 (t, 2H), 4.06 (s, 3H), 5.05 (m, 2H), 6.98(m, 1H), 8.16 (m, 1H), 8.50 (m, 1H). MS APCI+m/z 391 [MH]⁺.

Step 8 Methyl1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(6-ethylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate

A solution of N-ethylmethylamine (4.6 mL, 53.8 mmol) inN-methylpyrrolidin-2-one (7 mL) was added to a solution of themonochloro compound of step 7 (7.0 g, 17.93 mmol) inN-methylpyrrolidin-2-one (28 mL) at 110 C. The reaction mixture washeated overnight and on completion the solution was cooled to roomtemperature and water (25 mL) was added. After stirring at roomtemperature for 2 hours the slurry was filtered and washed with 2×15 mLwater. The solid was dried overnight in vacuo at 55 C to give an orangesolid (5.988 g, 15.0 mmol, 84%). ¹H NMR (CD₃OD, 400 MHz) δ: 1.12 (m,3H), 1.25 (m, 3H), 2.40 (s, 3H), 3.21 (m, 2H), 3.23 (s, 3H), 3.60 (m,2H), 3.75 (m, 2H), 3.96 (s, 3H), 4.80 (m, 2H), 6.94 (m, 1H), 8.16 (m,1H), 8.34 (m, 1H). MS APCI−m/z 412 [M-H]⁻.

Step 91-(2-Ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(6-ethylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylicAcid

The ester of step 8 (13.57 g, 32.83 mmol) and a 1M aqueous solution ofsodium hydroxide (90 mL) were dissolved in methanol (10 mL) and thereaction mixture stirred at 85 C for 1 hour. The reaction mixture wascooled to room temperature and acidified with 10% aqueous citric acid(90 mL). The aqueous layer was extracted twice with dichloromethane (36mL and 24 mL). The aqueous layer was further acidified with 10% aqueouscitric acid (20 mL) and extracted with dichloromethane (24 mL). Thecombined dichloromethane extracts were combined and ethanol (13 mL) wasadded. The solution was distilled at ambient pressure and the distilleddichloromethane replaced with ethanol (52 mL). Water (12 mL) was addedand the mixture was cooled to 5 C and stirred for 1 hour. The slurry wasfiltered and washed with water (24 mL) and dried in vacuo at 55 C togive a yellow solid (8.858 g, 22.2 mmol, 68%)

¹H NMR (CD₃OD, 400 MHz) δ: 1.10 (t, 3H), 1.30 (t, 3 H), 2.43 (s, 3H),3.24 (s, 3H), 3.57 (m, 2H), 3.70 (m, 2H), 3.93 (t, 2H), 4.84 (m, 2H),7.02 (m, 1H), 8.13 (m, 1H), 8.16 (m, 1H). MS APCI+m/z 400 [M-H]⁺.

Step 10N-[1-(2-Ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide

The carboxylic acid of step 9 (29.0 g, 72.6 mmol), methanesulphonamide(8.28 g, 87 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (18.0 g, 94 mmol) and 4-dimethylaminopyridine (10.59 g, 94mmol) were dissolved in dichloromethane (385 mL) and the reactionmixture stirred at room temperature overnight. The reaction mixture wasdiluted with dichloromethane (to 1500 mL) and washed twice with aqueouscitric acid (50 g in 200 mL), then washed once with an acidic solutionof a mixture of citric acid and sodium hydroxide. The dichloromethanephase was dried over magnesium sulphate and concentrated in vacuo. Thesolid residue was refluxed in isopropanol (1 L) for 20 minutes, allowedto cool and the resulting solid filtered off. The isolated yellow solidwas then refluxed in ethyl acetate (2000 mL) until solution occurred,whereupon the volume of ethyl acetate was reduced to 1000 mL. Theresulting solution was filtered and allowed to cool to room temperatureovernight and then placed in an ice bath and stirred for 1.5 hours. Theresulting solid was filtered off and washed with ether (2×50 ml), driedon the filter pad for 3 hours and then in vacuo over phosphoruspentoxide to yield a white powder (16.7 g). PXRD analysis of the powderindicated that it was the Form A crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

¹H NMR (CDCl₃, 400 MHz) δ: 1.20 (t, 3H), 1.29 (t, 3H), 2.41 (s, 3H),3.24 (s, 3H), 3.45 (s, 3H), 3.64 (q, 2H), 3.75 (m, 2H), 3.99 (t, 2H),4.82 (m, 2H), 6.87 (d, 1H), 8.20 (d, 1H), 8.29 (s, 1H), 9.87 (br, 1H).MS ES+m/z 477 [MH]⁺. Found C, 50.25: H, 5.90: N, 23.41: Calculated forC20H28N8O4S; C, 50.41: H, 5.92: N, 23.51.

Example 4 Preparation of Form A (Recrystallization From IsopropylAlcohol)

The Form A crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

N-[1-(2-Ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide

CrudeN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide(16.7 g) (see Example 1) was slurried in dichloromethane (20 mL) andisopropyl alcohol (70 mL). The slurry was heated to reflux (about 60°C.) and the solid material appeared to remain substantially undissolved.An additional amount of dichloromethane (40 mL) was added in 5 mLincrements to the slurry. The resulting solution was refluxed for aboutone minute and heating was stopped. At the end of this time the solidappeared to have dissolved to yield a yellow solution. The solution wasthen cooled to 35° C. with no sign of crystallization. The solution wasseeded with a small amount (less than about 0.5 g) of crudeN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewith no sign of crystallization. The solution was further cooled to roomtemperature with no sign of crystallization. When cooled to 5° C., thesolution became a slurry. This slurry was stirred at a similartemperature, then filtered and the material collected on the filter wasdried at 50° C. to yield a solid (7.7 g). PXRD analysis of the solidindicated that it was the Form A crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

Example 5 Preparation of Form B (Methanol Reflux)

The Form B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

N-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide(13.9 g) containing crystalline form Form A (see Example 3) wasdissolved in refluxing dichloromethane (160 mL) and methanol (200 mL).Dichloromethane was distilled out (approximately 110 mL distillatecollected). The mixture was cooled to room temperature, granulated for30 minutes, and filtered. The solids were washed with methanol (30 mL),and dried in vacuo to yield a bright yellow solid (10.8 g). PXRDanalysis of the solid indicated that it was the Form B crystalline formofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

Example 6 Preparation of Form B (Methanol Reflux)

The Form B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

1-(2-Ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylicacid (1.19 kg, 2.98 mole) (see Example 3, Step 9), methanesulphonamide(344 g, 3.6 mole), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (810.0 g, 4.21 mole), and 4-dimethylaminopyridine (488.8g, 4.01 mole) were dissolved in dichloromethane (12 L) under a nitrogenatmosphere and the reaction mixture stirred at room temperature. After 3hours, to the solution was added a further portion of4-dimethylaminopyridine (62.0 g, total 551.7 g, 4.52 mole) and thereaction mixture stirred at room temperature for a further 20 hours. Thereaction mixture was diluted with 10% aqueous citric acid (12 L) and theorganic phase was separated, washed with 10% aqueous citric acid (12 L),and then washed with water.

The resulting solution (10 L) was filtered and was distilled atatmospheric pressure to approximately half of its initial volume, andthe hot solution was diluted with portions of methanol (total 14 L)whilst dichloromethane was removed in portions by distillation(distillate fractions totaling 11 L, giving a final volume of 13 L whichrefluxes at 55° C.). The yellow slurry was cooled to room temperature,stirred overnight, and then cooled to 5° C. The slurry was then filteredand washed with chilled methanol portions (totaling 5.8 L). The materialcollected from the filter was dried in vacuum at 55° C. for 3 days togive the product as a bright yellow solid (1.038 kg, 73% yield) that itwas the Form B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

Example 7 Preparation of Form B (Dexoygenation and Methanol Reflux)

The Form B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

Dichloromethane (260 mL) was refluxed with a flow of nitrogen throughthe vessel headspace, reducing the volume to 240 mL, and then cooled toroom temperature under a nitrogen atmosphere.1-(2-Ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(6-ethylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylicacid (24 g, 60 mmole) (see Example 3, Step 9), methanesulphonamide (6.88g, 72 mmole), and 4-dimethylaminopyridine (10.98 g, 90 mmole) weredissolved in the dichloromethane (240 mL) under a nitrogen atmosphere.The solution was stirred for 30 minutes then1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.13 g, 84mmole) was added and the reaction mixture was stirred at roomtemperature overnight under a nitrogen atmosphere. The reaction mixturewas diluted with 10% aqueous citric acid (240 mL) and the organic phasewas separated, washed with 10% aqueous citric acid (240 mL), and thenwashed with water (240 mL).

The resulting solution was distilled at atmospheric pressure toapproximately half of its initial volume (approximately 120 mL). The hotsolution was slowly diluted with methanol (240 mL) and then the mixturewas distilled at atmospheric pressure to approximately 240 mL. The hotmixture was again diluted with methanol (120 mL), and again distilled atatmospheric pressure to approximately 240 mL. The hot mixture was yetagain diluted with methanol (120 mL), and yet again distilled atatmospheric pressure to approximately 240 mL. The mixture was allowed tocool to room temperature with stirring over one hour, and then cooledand stirred at 0-5° C. for 1 hour. The resulting yellow slurry was thenfiltered and the solids washed with chilled methanol (96 mL). The solidswere dried in vacuum overnight at 55° C. to give a bright yellow solid(25.78 g, 90% yield) that it was the Form B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

Example 8 Preparation of Form B (Ion Exchange Resin and Methanol Reflux)

The Form B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

1-(2-Ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(6-ethylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylicacid (24 g, 60.1 mmole) (see Example 3, Step 9), methanesulphonamide(6.88 g, 72.4 mmole), and 4-dimethylaminopyridine (10.98 g, 90 mmole)were dissolved in dichloromethane (240 mL) under a nitrogen atmosphere.The solution was stirred for 30 minutes then1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.14 g,84.1 mmole) was added and the reaction mixture was stirred at roomtemperature until the reaction was judged essentially complete after 5hours. The reaction mixture was diluted with 10% aqueous citric acid(240 mL) and the organic phase was separated, washed with 10% aqueouscitric acid (240 mL), and then washed with water (240 mL).

To the stirred separated organic phase was added Amberlite IRN-78 (24 g)(a basic ion-exchange resin) and the mixture was stirred for 3 hours.The resin beads were filtered of, the filter cake was washed withdichloromethane (48 mL), and the combined filtrates were washed with 10%aqueous citric acid (120 mL), and then twice washed with water (240 mL).

The resulting solution was distilled at atmospheric pressure toapproximately half of its initial volume (approximately 120 mL). The hotsolution was slowly diluted with methanol (240 mL), precipitating ayellow solid, and then the mixture was distilled at atmospheric pressureto approximately 240 mL. The hot mixture was again diluted with methanol(240 mL), and again distilled at atmospheric pressure to approximately240 mL. The yellow slurry was allowed to cool to room temperature withstirring overnight, and then cooled in an ice bath for 1 hour (toapproximately 0-5° C.). The resulting slurry was then filtered and thesolids washed with methanol (96 mL). The solids were dried in vacuumovernight at 50° C. to give a bright yellow solid (21.51 g, 75.1% yield)that it was the Form B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

Example 9 Preparation of Form B (Slurry Conversion)

Approximately 25 mg of Form A (see Example 3) was slurried at roomtemperature with 1 ml of methanol. There was a rapid increase in theyellow color of the slurry within 10 minutes. A small sample was removedfrom the slurry. PXRD analysis of the sample indicates that it was theForm B crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.

Example 10 Preparation of Form B (Temperature Conversion)

A sample of Form A (see Example 3) was heated to 180° C. using DSC. Thesample melts and recrystallizes as Form B. The sample was allowed tocool to room temperature. To confirm that no Form A remained and thatthe conversion to Form B was complete, the sample was heated again inthe DSC to 175° C. No significant thermal events were detected. Thesample was allowed to cool to room temperature. The sample was heatedagain to 250° C. The melt of form B was observed at 220° C.

Example 11 Preparation of Form C (Slurry Conversion)

The Form C crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

A sample of Form B (see Examples 5 to 9) was slurried in THF/H₂O (50:50volume/volume) at 4° C. After 16 days a small sample was filtered anddried at room temperature, resulting in a light yellow solid. DSCanalysis of the sample was consistent with Form C. Another small samplewas removed in the wet state from the slurry. PXRD analysis of thesample was consistent with Form C. After 31 days of slurrying, anothersmall sample was removed in the wet state from the slurry. PXRD analysisof the sample was consistent with Form C.

Example 12 Preparation of Form C (Seeding)

The Form C crystalline form ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidewas prepared as follows:

A sample of Form B (see Examples 5 to 9) (129.6 g) was stirred inacetone (1300 ml) at 23° C. under an atmosphere of nitrogen. A seed ofForm C (20 mg) was added, and stirring was continued for 13 days atambient temperature. The solids were collected by filtration, and driedunder vacuum at ambient temperature but above 20° C., to provide a 92.4%yield of the product Form C (119.8 g), displaying the powder X-raydiffraction pattern in FIG. 4.

Example 13 Stability of Form A and Form B

The thermodynamically stabilities of Form A and Form B were compared asdescribed below. First, approximately 25 mg of Form A was slurried inapproximately 1 ml of methanol. There was a rapid increase in the yellowcolor of the slurry. A small sample was removed from the slurry. PXRDanalysis confirmed that the sample was Form B. Second, Form A and Form Bwere analyzed by DSC and illustrative data for samples of Form A andForm B are shown in FIGS. 5 and 6, respectively. The DSC data indicatedthat the melting point of Form B was higher than that of Form A.Accordingly, the results of both the slurry conversion analysis and theDSC analysis confirm that Form B is more thermodynamically stable thanForm A.

Example 14 Stability of Form B and Form C

Bridging studies were performed in the following solvent systems todetermine the relative thermodynamic stabilities of Form B and Form C:(1) THF/H₂O (50:50 volume/volume), (2) methyl ethyl ketone (“MEK”), (3)methanol, and (4) methanol/dichloromethane (DCM) (50:50 volume/volume).The MEK study was performed at room temperature while the studies withthe other three solvent systems were performed at both 40° C. and 60° C.In each study a suspension of Form B in the appropriate solvent systemwas prepared. Approximately 10 mg of Form C was then added to eachsuspension. The suspensions were then allowed to slurry at theappropriate temperature for a specific period of time. The solventsystem, temperature of the solvent system, and period of time over whichthe suspension was allowed to slurry are set forth below for each study:

(1) Study A: Slurried for 3 days in MEK at room temperature;(2) Study B: Slurried for 3 days in THF/H₂O (50:50) at 40° C.;(3) Study C: Slurried for 3 days in THF/H₂O (50:50) at 60° C.;(4) Study D: Slurried for 21 days in Methanol at 40° C.;(5) Study E: Slurried for 21 days in Methanol at 60° C.;(6) Study F: Slurried for 5 days in Methanol/DCM at 40° C.; and(7) Study G: Slurried for 21 days in Methanol/DCM at 60° C.

At the end of the specified time period, a sample was removed from eachslurry and analyzed by PXRD. The PXRD analyses indicated that all of thesamples produced at room temperature and at 40° C. were Form C and allof the samples produced at 60° C. were Form B. As discussed previously,there is a crossover in the thermodynamic stability of Form B and Form Cat a temperature between 40 and 60° C. (i.e. the two forms areenantiotropes). At temperatures below this crossover point, Form C isthe most thermodynamically stable form. At temperatures above thiscrossover point, Form B is the most thermodynamically stable form.

Example 15 Ex Vivo Assays Method A: Aortic Ring Assay

This protocol describes a procedure for measuring the direct relaxationof rat aortic rings exposed toN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.In this assay, PDE5 inhibiting compounds elicit a relaxation of aorticrings by enhancing the cGMP signal evoked by a stable exogenousNO-donor, Diethyltriamine NONOate (diazen-1-ium-1,2-diolate)(“DETA-NO”). An EC₅₀, with 95% confidence intervals, for compound-evokedrelaxation is calculated as an index of potency. The EC₅₀ is theconcentration of the PDE5 inhibiting compound which produces 50% of themaximum possible effective response for the PDE5 inhibiting compound.

Male Sprague-Dawley rats (250-350 g) are asphyxiated by CO₂ gas andtheir thoracic aorta carefully excised and placed in Krebs buffer. Theaortas are then carefully dissected free of connective tissue anddivided into 8 sections, each 3-4 mm in length.

Aortic rings are suspended between parallel stainless steel wires in awater jacketed (37° C.), 15 mL tissue bath under a resting tension of 1gram. Tension is measured using isometric tension transducers andrecorded using Ponemah tissue platform system. Each preparation isallowed to equilibrate for at least 60 minutes prior to drug testing.During this time, the tissues are also incubated with 200 uMNG-monomethyl L-arginine (“L-NMMA”), and the incubation media changedevery 15-20 minutes (L-NMMA is added after each wash to maintain thefinal concentration at 200 uM in each tissue bath).

Following the equilibration period, baseline tensions are recorded foreach tissue. The vasoconstrictor response to phenylepherine (1 uM) isassessed and when the response to phenylepherine reached a maximum,vascular reactivity was subsequently assessed by a challenge ofacetylcholine (1 uM). Following another washout period, a secondbaseline value is recorded, the vasoconstrictor noradrenaline (25 nM) isadded to each bath and the tissues incubated for a time period (about 15minutes) sufficient for the tissues to achieve a stable tone. Anexogenous NO drive is supplied using the stable NO-donor, DETA-NO. Theconcentration of DETA-NO is titrated (cumulatively in half-logincrements) to achieve approximately 5-15% relaxation of thenoradrenaline-evoked preconstriction. Cumulative concentration-responsecurves are constructed in a single ring, typically using 5 doses/ringand allowing 15 minutes between each addition.

Method B: Aortic Ring Assay

The protocol of Method A can be modified to provide an alternativeprotocol to measure the relaxation of rat aortic rings exposed to PDE5inhibiting compounds. This alternative method varies from Method A asdescribed below:

For the alternative method, the endothelium is first removed by gentlyrubbing the lumen of the vessel together between the fingers prior topreparing the rings (denuded rings). The resting tension is set at 2grams and the vasoconstrictor response to a maximal concentration ofphenylepherine (1 μM) is assessed, followed (after a washout period) bytwo further exposures to 300 nM of phenylephrine. Theconcentration-response relationship to noradrenaline is constructed ineach tissue over concentration range 0.1 to 300 nM. After anotherwashout period, the tissues are constricted with an EC₉₀ concentrationof noradrenaline for compound testing.

Example 16 In Vivo Assays Method A: Culex™ Assay

A conscious pre-cannulated spontaneously hypertensive rat (SHR) model isused for evaluating the efficacy ofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidein lowering systemic arterial blood pressure. This assay is conductedusing an automated blood sampler (“ABS”) system. The Culex™ ABS system(Bioanalytical System, Inc., West Lafayette, Ind.) comprises a laptopcomputer, four control units and metabolic cages. This ABS system allowsfor the collection of multiple blood samples from a single rat withoutcausing undue stress to the animal.

In addition, the ABS system allows for the collection of urine samplesthat can be potentially used for biomarker identifications. Through thisapproach, efficacy and standard pharmacokinetic studies are conducted inthe conscious unrestrained SHR rats simultaneously to define therelationship between plasma free drug concentration or potentialbiomarker(s) and pharmacological effect (reduction of mean arterialblood pressure).

SHR rats at 12 to 16 weeks of age, weighing about 300 g, undergosurgerical cannulation of both jugular veins and the right carotidartery. After surgical recovery, animals are placed in the Culex™ cagesand tethered to a movement-responsive arm with a sensor that controlscage movement when the animal moves to prevent the catheters from beingtwisted. Connections are made between the right jugular catheter and theCulex™ sterile tubing set for blood sampling, and the left jugularcatheter for compound administration, and the catheter in the rightcarotid artery is connected to a pressure transducer for monitoringblood pressure. To keep the patency of the catheters, the right jugularcannula is maintained by the “tend” function of the Culex™ that flushesthe catheter with 20 μL heparin saline (10 units/mL) every 12 minutes orbetween sampling events, and the left jugular cannula is filled withheparin saline (20 units/mL). The patency of the right carotid cannulais maintained by slow infusion of heparin saline either directly intothe extend tubing when blood pressure is not recorded or through thepressure transducer during the blood pressure monitoring. Animals areallowed to acclimate for at least two hours before compound evaluation.The PDE5 inhibiting compounds may be administered intravenously or byoral gavage. Blood sampling protocols (sampling time and volume) areprogrammed using the Culex™ software. The total amount of bloodwithdrawn from each animal will not exceed 750 μL/24 hrs and 10 mL/kgwithin two weeks. Heart rate, blood pressure, and drug concentration aremonitored. Systemic arterial blood pressure and heart rate are recordedby PONEMAH (Gould Instrument System, Valley View, Ohio), a pressuretransducer through a data acquisition system for recording bloodpressure and heart rate, for 6 to 24 hours based on experimentalprotocol. Mean arterial blood pressure (primary endpoint) is analyzedfor assessing the efficacy of the compound.

Blood samples are analyzed for measuring plasma drug concentration,using the LC/MS/MS method described below, and for evaluating potentialbiomarkers.

LC/MS/MS Method

Sample Preparation: Plasma samples (50 μL unknown, control or blank) aremixed with 10 μL acetonitrile:water or a standard solution of the PDE-5inhibiting compound and 150 μL of internal standard solution (100 ng/mLof the PDE-5 inhibiting compound in acetonitrile). The mixture iscentrifuged at 3000 rpm for 5 min, and 125 μL of the supernatanttransferred to a 96 well plate. The solvent is evaporated under a streamof nitrogen and the residue is reconstituted with 80 μLacetonitrile/0.1% aqueous formic acid (20:80 v/v).

A 20 μL volume of each prepared sample is injected onto a PhenomenexSynergi 4 μm MAX-RP 2.0×75 mm column and eluted at 0.4 mL/min usinggradient elution from 0.1% aqueous formic acid (mobile phase A) toacetonitrile (mobile phase B). The gradient program consists of initialapplication of 90% mobile phase A, followed by a linear gradient to 75%mobile phase B from 0.2 to 1.15 min after injection and held at 75%mobile phase B until 2.0 min. The mobile phase was linearly changed backto 90% mobile phase A from 2.00 to 2.10 minutes, and the next injectiontook place at 3.00 min. Detection was performed by mass spectrometryusing positive ion electrospray (ESI) with multiple reaction monitoringof the transitions m/z 454.00 (MH+ the PDE-5 inhibiting compound)→m/z408.00, m/z 466.24 (MH+ the PDE-5 inhibiting compound)→409.33. The ionspray voltage a is set at 5000. A calibration curve is constructed byusing peak area ratios of the analyte relative to the internal standard.Subject concentrations are determined by inverse prediction from theirpeak area ratios against the calibration curve.

Method B: Implantation of Radio Transmitters and Subsequent BloodPressure Screening by Telemetry in Spontaneously Hypertensive Rats

Spontaneously Hypertensive Rats (SHR) are anesthetized with isofluranegas via an isoflurane anesthesia machine that is calibrated to deliverisoflurane over a range of percentages as oxygen passes through themachine's inner chambers. The animals are placed in an induction chamberand administered isoflurane at 4-5% to reach a surgical plane ofanesthesia. They are then maintained at 1-2% during the surgicalprocedure via a nose cone, with isoflurane delivered via a smallerisoflurane anesthesia device on the surgical table.

Following administration of anesthesia, the rats are implanted withtransmitters using aseptic procedures with commercially availablesterile radio-telemetry units (Data Sciences, international, Roseville,Minn. 55113-1136). Prior to surgery the surgical field is shaved,scrubbed with Dial™ brand antimicrobial solution (containing 4%chlorhexidine gluconate and 4% isopropyl alcohol) followed by anapplication of iodine (10%) spray solution. A 2.5 to 3.0 cm laparotomyis preformed and the radio-telemetry units implanted into the abdomen,with the catheter tip inserted into the abdominal aorta. Baby Weitlanerretractors are used to retain soft tissue. A 1 cm section of theabdominal aorta is partially dissected and that section cross-clampedbriefly, punctured with a 21-gauge needle and the transmitter cathetertip introduced into the vessel and secured by a single 4.0 silk sutureanchored to the adjacent psoas muscle. The transmitter body is theninserted into the abdominal cavity and simultaneously secured to theabdominal muscle wall while closing with running 4.0 silk suture. Theskin layer is closed with subdermal continuous 4.0 absorbable suture. Asubcutaneous (s.c.) administration of marcaine followed by a topicalapplication of iodine is administered into and around the suture line,respectively, upon closing. All rats receive a postoperative injectionof buprenorphine @ 0.05 mg/kg, s.c. before regaining consciousness. Atypical dose volume for a 0.300 kg rat will be 0.050 ml. The rats mustbe fully recovered from their operative anesthesia before theadministration of buprenorphine. They then receive the same dose oncedaily for 2 consecutive days, unless the animal demonstrates that it isin compromising postoperative pain.

Following surgery, the rats are returned to their cages and housedindividually on solid bottom caging with paper bedding. A period of noless than 7 days is allowed for recovery before experimental proceduresare initiated. It has been observed that the rats are typicallyhypertensive for several days following surgery and return to“normotensive” levels by approximately the 7^(th) day post-surgery. Theyare fed standard rat chow and water ad libitum throughout theexperimental time line.

The compound is administered intragastrically (i.g.) via gavage, usingof a stainless steel, 2½ inch, 18 gauge gavage needle with a balled end.For single daily dosing, the target volume is 3.33 ml/kg, i.g. Thevehicles in which the compound is administered will vary depending onsolubility of the compound, however, methylcellulose (0.5%) in waterwill be the primary choice.

Blood pressure data will be obtained using Data Sciences International'sdata acquisition program. Blood pressure samples are recorded at 1.5-3minute intervals for a 5 second duration 24 hours per day for the entirestudy. This data is processed by Data Science's data analysis softwareinto averages of a desired time intervals. All other data reduction isperformed in Microsoft Excel™ spreadsheets.

Method C: SHR Rat

This experimental protocol is designed to screen for blood pressurelowering byN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide.The spontaneously hypertensive rat (SHR) is cannulated in the jugularvein and carotid artery; one for compound administration and one fordirect blood pressure measurement, respectively. The animals are fullyconscious following surgery and all experimentation takes place withinone working day. Blood pressure lowering is the primary parameter to beevaluated. However, systolic and diastolic pressure and heart rate datais collected as well. Rats will be dosed in an escalating, or cumulativemanner to observe the responses following this regimen. This particularmethod will permit screening of multiple doses in one day using the sameanimals.

Methods:

Anesthesia: Rats are anesthetized with 5% isoflurane to effect. Incisionsites are shaved and aseptically prepared for surgery. Rats are thentransferred to the surgical field with a heating pad, supplementalisoflurane and maintained at 37° C., and isoflurane to effect throughoutthe surgical procedure.

Surgery: Arterial and venous cannula are implanted in the jugular veinand carotid artery, respectively. Cannulae are tunneled subcutaneouslyto the back of the neck where they exit percutaneously. Stainless steelstaples are used to close each incision site. The cannulae are then runthrough a spring-tether to a swivel apparatus by which protects thecannulae from the animals chewing throughout the experiment.

Recovery: Rats are placed into an opaque polycarbonate cage instrumentedwith a counter balance arm that supports the weight of the tether andswivel apparatus. A paper bedding material is used to cover the bottomof the cage. The rats are allowed to recover from surgery at this pointand receive 2 mL of volume early during their recovery stage. No food isprovided to the animals.

When introducing elements of the present invention or the exemplaryembodiment(s) thereof, the articles “a,” “an,” “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising,” “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements. Although this invention has been described with respect tospecific embodiments, the details of these embodiments are not to beconstrued as limitations.

1. CrystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamidehaving an X-ray powder diffraction pattern comprising diffraction peaksat 8.5±0.1, 9.0±0.1, and 21.0±0.1 degrees two theta.
 2. The crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 1 wherein the X-ray powder diffraction pattern furthercomprises diffraction peaks at 14.0±0.1, 16.9±0.1, and 19.9±0.1 degreestwo theta.
 3. The crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 2 wherein the X-ray powder diffraction pattern furthercomprises diffraction peaks at 21.4±0.1, 21.7±0.1, and 22.5±0.1 degreestwo theta.
 4. The crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 1 wherein the X-ray powder diffraction pattern furthercomprises diffraction peaks as depicted in FIG.
 1. 5. The crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 1 having a Fourier Transform infrared spectrum comprising anabsorption band at 3247±2 cm⁻¹.
 6. The crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 5 wherein the Fourier Transform infrared spectrum furthercomprises at least one absorption band selected from the groupconsisting of 696±2, 1085±2, 1188±2, and 1540±2.
 7. The crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 1 having a Raman spectrum comprising a band at 3255±3 cm⁻¹. 8.The crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 7 wherein the Raman spectrum further comprises at least oneband selected from the group consisting of 993±2, 1383±2, 1473±2, and1569±2 cm⁻¹.
 9. A pharmaceutical composition comprising atherapeutically effective amount of the crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 1, and a pharmaceutically-acceptable carrier.
 10. Apharmaceutical composition comprising a therapeutically effective amountofN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3d]pyrimidine-3-carbonyl]methanesulfonamideand a pharmaceutically-acceptable carrier, wherein at least about 50weight percent of theN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-yl-amino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideis present as the crystallineN-[1-(2-ethoxyethyl)-5-(N-ethyl-N-methylamino)-7-(4-methylpyridin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamideof claim 1.